Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
Agonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulationAgonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulation
Agonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulationAgonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulation
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as potentiation of PGE2-induced cAMP accumulation by scintillation proximity assayAgonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as potentiation of PGE2-induced cAMP accumulation by scintillation proximity assay
Agonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as potentiation of PGE2-induced cAMP accumulation by scintillation proximity assayAgonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as potentiation of PGE2-induced cAMP accumulation by scintillation proximity assay
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Agonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as potentiation of PGE2-induced cAMP accumulation by scintillation proximity assayAgonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as potentiation of PGE2-induced cAMP accumulation by scintillation proximity assay
Agonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as potentiation of PGE2-induced cAMP accumulation by scintillation proximity assayAgonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as potentiation of PGE2-induced cAMP accumulation by scintillation proximity assay
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulationAgonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulation
Agonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulationAgonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulation
Agonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF methodAgonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF method
Agonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF methodAgonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF method
Agonist activity at rat EP4 receptor expressed in HEK293 cells assessed as cAMP activationAgonist activity at rat EP4 receptor expressed in HEK293 cells assessed as cAMP activation
Agonist activity at rat EP4 receptor expressed in HEK293 cells assessed as cAMP activationAgonist activity at rat EP4 receptor expressed in HEK293 cells assessed as cAMP activation
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity against rat EP4 receptor expressed in HEK293 cells assessed as stimulation of cAMP releaseAgonist activity against rat EP4 receptor expressed in HEK293 cells assessed as stimulation of cAMP release
Agonist activity against rat EP4 receptor expressed in HEK293 cells assessed as stimulation of cAMP releaseAgonist activity against rat EP4 receptor expressed in HEK293 cells assessed as stimulation of cAMP release
Agonist activity against rat EP4 receptor expressed in HEK293 cells assessed as stimulation of cAMP releaseAgonist activity against rat EP4 receptor expressed in HEK293 cells assessed as stimulation of cAMP release
Agonist activity against rat EP4 receptor expressed in HEK293 cells assessed as stimulation of cAMP releaseAgonist activity against rat EP4 receptor expressed in HEK293 cells assessed as stimulation of cAMP release
Agonist activity against rat EP4 receptor expressed in HEK293 cells assessed as stimulation of cAMP releaseAgonist activity against rat EP4 receptor expressed in HEK293 cells assessed as stimulation of cAMP release
Agonist activity against rat EP4 receptor expressed in HEK293 cells assessed as stimulation of cAMP releaseAgonist activity against rat EP4 receptor expressed in HEK293 cells assessed as stimulation of cAMP release
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as potentiation of PGE2-induced cAMP accumulation by scintillation proximity assayAgonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as potentiation of PGE2-induced cAMP accumulation by scintillation proximity assay
Agonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as potentiation of PGE2-induced cAMP accumulation by scintillation proximity assayAgonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as potentiation of PGE2-induced cAMP accumulation by scintillation proximity assay
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: EP4 receptors couple to Gs and mediate elevations in cAMP concentration, although they do participate in other pathways as well. There are some redundancies in function between EP2 and EP4 receptors. For example, both receptors induce PGE2-mediated RANKL through cAMP.cAMP Assay: EP4 receptors couple to Gs and mediate elevations in cAMP concentration, although they do participate in other pathways as well. There are some redundancies in function between EP2 and EP4 receptors. For example, both receptors induce PGE2-mediated RANKL through cAMP.
cAMP Assay: EP4 receptors couple to Gs and mediate elevations in cAMP concentration, although they do participate in other pathways as well. There are some redundancies in function between EP2 and EP4 receptors. For example, both receptors induce PGE2-mediated RANKL through cAMP.cAMP Assay: EP4 receptors couple to Gs and mediate elevations in cAMP concentration, although they do participate in other pathways as well. There are some redundancies in function between EP2 and EP4 receptors. For example, both receptors induce PGE2-mediated RANKL through cAMP.
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at mouse EP4 receptor expressed in CHO cells assessed as cAMP productionAgonist activity at mouse EP4 receptor expressed in CHO cells assessed as cAMP production
Agonist activity at mouse EP4 receptor expressed in CHO cells assessed as cAMP productionAgonist activity at mouse EP4 receptor expressed in CHO cells assessed as cAMP production
Agonist activity at mouse EP4 receptor expressed in CHO cells assessed as cAMP productionAgonist activity at mouse EP4 receptor expressed in CHO cells assessed as cAMP production
Agonist activity at mouse EP4 receptor expressed in CHO cells assessed as cAMP productionAgonist activity at mouse EP4 receptor expressed in CHO cells assessed as cAMP production
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.
Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Agonist activity at human recombinant EP4 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP4 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay
Agonist activity at human recombinant EP4 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP4 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay
Agonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulationAgonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulation
Agonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulationAgonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulation
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Agonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay
Agonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.
Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Agonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF methodAgonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF method
Agonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF methodAgonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF method
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Agonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulationAgonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulation
Agonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulationAgonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulation
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Agonist activity at rabbit EP4 receptor assessed as relaxation of Kcl-induced tissue contraction by isometric transducer methodAgonist activity at rabbit EP4 receptor assessed as relaxation of Kcl-induced tissue contraction by isometric transducer method
Agonist activity at rabbit EP4 receptor assessed as relaxation of Kcl-induced tissue contraction by isometric transducer methodAgonist activity at rabbit EP4 receptor assessed as relaxation of Kcl-induced tissue contraction by isometric transducer method
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulationAgonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulation
Agonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulationAgonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulation
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Agonist activity at human recombinant EP4 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP4 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay
Agonist activity at human recombinant EP4 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP4 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Agonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay
Agonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Agonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulationAgonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulation
Agonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulationAgonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulation
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Agonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF methodAgonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF method
Agonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF methodAgonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF method
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay
Agonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay
Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.
Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.
Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Agonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulationAgonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulation
Agonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulationAgonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulation
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Agonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay
Agonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells