Agonist activity at human EP1 expressed in HEK293 cells assessed as increase in cAMP level incubated for 20 mins measured after 60 mins addition of cAMP detect reagent by HTRF analysisAgonist activity at human EP1 expressed in HEK293 cells assessed as increase in cAMP level incubated for 20 mins measured after 60 mins addition of cAMP detect reagent by HTRF analysis

Agonist activity at human EP1 expressed in HEK293 cells assessed as increase in cAMP level incubated for 20 mins measured after 60 mins addition of cAMP detect reagent by HTRF analysisAgonist activity at human EP1 expressed in HEK293 cells assessed as increase in cAMP level incubated for 20 mins measured after 60 mins addition of cAMP detect reagent by HTRF analysis

Agonist activity at human EP1 expressed in HEK293 cells assessed as increase in cAMP level incubated for 20 mins measured after 60 mins addition of cAMP detect reagent by HTRF analysisAgonist activity at human EP1 expressed in HEK293 cells assessed as increase in cAMP level incubated for 20 mins measured after 60 mins addition of cAMP detect reagent by HTRF analysis

Agonist activity at human EP1 expressed in HEK293 cells assessed as increase in cAMP level incubated for 20 mins measured after 60 mins addition of cAMP detect reagent by HTRF analysisAgonist activity at human EP1 expressed in HEK293 cells assessed as increase in cAMP level incubated for 20 mins measured after 60 mins addition of cAMP detect reagent by HTRF analysis

Agonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human recombinant EP1 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP1 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP1 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP1 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=100%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=100%)

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=100%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=100%)

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=100%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=100%)

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.

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 recombinant human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level measured at 3 secs time interval by fura-2-AM dye based fluorescence assayAgonist activity at recombinant human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level measured at 3 secs time interval by fura-2-AM dye based fluorescence assay

Agonist activity at recombinant human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level measured at 3 secs time interval by fura-2-AM dye based fluorescence assayAgonist activity at recombinant human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level measured at 3 secs time interval by fura-2-AM dye based fluorescence assay

Agonist activity at human recombinant EP1 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP1 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP1 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP1 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human EP1 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP1 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay

Agonist activity at human EP1 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP1 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay

Agonist activity at human EP1 expressed in HEK293 cells assessed as increase in cAMP level incubated for 20 mins measured after 60 mins addition of cAMP detect reagent by HTRF analysisAgonist activity at human EP1 expressed in HEK293 cells assessed as increase in cAMP level incubated for 20 mins measured after 60 mins addition of cAMP detect reagent by HTRF analysis

Agonist activity at human EP1 expressed in HEK293 cells assessed as increase in cAMP level incubated for 20 mins measured after 60 mins addition of cAMP detect reagent by HTRF analysisAgonist activity at human EP1 expressed in HEK293 cells assessed as increase in cAMP level incubated for 20 mins measured after 60 mins addition of cAMP detect reagent by HTRF analysis

Agonist activity at human EP1 expressed in HEK293 cells assessed as increase in cAMP level incubated for 20 mins measured after 60 mins addition of cAMP detect reagent by HTRF analysisAgonist activity at human EP1 expressed in HEK293 cells assessed as increase in cAMP level incubated for 20 mins measured after 60 mins addition of cAMP detect reagent by HTRF analysis

Agonist activity at human EP1 expressed in HEK293 cells assessed as increase in cAMP level incubated for 20 mins measured after 60 mins addition of cAMP detect reagent by HTRF analysisAgonist activity at human EP1 expressed in HEK293 cells assessed as increase in cAMP level incubated for 20 mins measured after 60 mins addition of cAMP detect reagent by HTRF analysis

Agonist activity at human EP1 expressed in HEK293 cells assessed as increase in cAMP level incubated for 20 mins measured after 60 mins addition of cAMP detect reagent by HTRF analysisAgonist activity at human EP1 expressed in HEK293 cells assessed as increase in cAMP level incubated for 20 mins measured after 60 mins addition of cAMP detect reagent by HTRF analysis

Agonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=100%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=100%)

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=100%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=100%)

Agonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

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.

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)

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 recombinant EP1 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP1 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP1 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP1 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at recombinant human EP1 receptor expressed in HEK293-EBNA cells co-expressing Gqs5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assayAgonist activity at recombinant human EP1 receptor expressed in HEK293-EBNA cells co-expressing Gqs5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assay

Agonist activity at recombinant human EP1 receptor expressed in HEK293-EBNA cells co-expressing Gqs5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assayAgonist activity at recombinant human EP1 receptor expressed in HEK293-EBNA cells co-expressing Gqs5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM 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.

Agonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP1 (% of control ligand, 17-phi-PGE2=80%)

Agonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP1 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Antagonist activity at EP1 receptor (unknown origin) by reporter gene assayAntagonist activity at EP1 receptor (unknown origin) by reporter gene assay

Antagonist activity at EP1 receptor (unknown origin) by reporter gene assayAntagonist activity at EP1 receptor (unknown origin) by reporter gene assay

Antagonist activity at EP1 receptor (unknown origin) by reporter gene assayAntagonist activity at EP1 receptor (unknown origin) by reporter gene assay

Antagonist activity at EP1 receptor (unknown origin) by reporter gene assayAntagonist activity at EP1 receptor (unknown origin) by reporter gene assay

Antagonist activity at EP1 receptor (unknown origin) by reporter gene assayAntagonist activity at EP1 receptor (unknown origin) by reporter gene assay

Antagonist activity at EP1 receptor (unknown origin) by reporter gene assayAntagonist activity at EP1 receptor (unknown origin) by reporter gene assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at EP1 receptor (unknown origin) by reporter gene assayAntagonist activity at EP1 receptor (unknown origin) by reporter gene assay

Antagonist activity at EP1 receptor (unknown origin) by reporter gene assayAntagonist activity at EP1 receptor (unknown origin) by reporter gene assay

Antagonist activity at EP1 receptor (unknown origin) by reporter gene assayAntagonist activity at EP1 receptor (unknown origin) by reporter gene assay

Antagonist activity at EP1 receptor (unknown origin) by reporter gene assayAntagonist activity at EP1 receptor (unknown origin) by reporter gene assay

Antagonist activity at EP1 receptor (unknown origin) by reporter gene assayAntagonist activity at EP1 receptor (unknown origin) by reporter gene assay

Antagonist activity at EP1 receptor (unknown origin) by reporter gene assayAntagonist activity at EP1 receptor (unknown origin) by reporter gene assay

Antagonist activity at EP1 receptor (unknown origin) by reporter gene assayAntagonist activity at EP1 receptor (unknown origin) by reporter gene assay

Antagonist activity at EP1 receptor (unknown origin) by reporter gene assayAntagonist activity at EP1 receptor (unknown origin) by reporter gene assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at human EP1 receptor assessed as inhibition of PGE-induced intracellular Ca2+ release at 1 uM by cell-based assayAntagonist activity at human EP1 receptor assessed as inhibition of PGE-induced intracellular Ca2+ release at 1 uM by cell-based assay

Antagonist activity at human EP1 receptor assessed as inhibition of PGE-induced intracellular Ca2+ release at 1 uM by cell-based assayAntagonist activity at human EP1 receptor assessed as inhibition of PGE-induced intracellular Ca2+ release at 1 uM by cell-based assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity human EP1 receptor assessed as inhibition of PGE2-induced intracellular Ca2+ release by cell-based assayAntagonist activity human EP1 receptor assessed as inhibition of PGE2-induced intracellular Ca2+ release by cell-based assay

Antagonist activity human EP1 receptor assessed as inhibition of PGE2-induced intracellular Ca2+ release by cell-based assayAntagonist activity human EP1 receptor assessed as inhibition of PGE2-induced intracellular Ca2+ release by cell-based assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assayAntagonist activity at rat His-tagged EP1 receptor expressed in African green monkey COS1 cells assessed as reduction in PGE2-induced increase in intracellular calcium level preincubated for 60 secs followed by PGE2 addition measured for 60 secs by Fluo 4-AM dye-based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Fluo-4 NW Calcium Assay Kit (Molecular Probes). The intracellular calcium concentration was measured as a fluorescent signal using FlexStation (registered trademark) (manufactured by Molecular Devices). 50 μL of each test compound that had been diluted with the assay buffer (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as decrease in prostaglandin E2-induced increase in intracellular calcium level incubated for 90 mins measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Antagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assayAntagonist activity at rat EP1 receptor expressed in African green monkey COS1 cells assessed as inhibition of prostaglandin-E2-induced increase in intracellular calcium level preincubated for 60 secs followed by prostaglandin-E2 addition measured for 60 secs by Fluo 4-AM dye based fluorescence assay

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.Cell Response Assay: The rat EP1 receptor-expressing cells were washed with the assay buffer. 100 μL of a fluorescent calcium indicator (Calcium kit II, Fluo 4 (Dojindo Laboratories).The intracellular calcium concentration was measured as a fluorescent signal using FDSS (registered trademark) 7000 (manufactured by Hamamatsu Photonics K. K.). 50 μL of each test compound (final concentrations: 1 nM to 10 μM) was added to each well after 20 seconds from initiating the reading of the fluorescent signal, and the fluorescence signal was measured for 60 seconds. Then, 50 μL of a prostaglandin E2 buffer solution was added to each well (final concentration 10 nM) and the fluorescence signal was measured for 60 seconds.

Functional activity in RAT-1 cells, transiently transfected with human prostaglandin EP1 receptorFunctional activity in RAT-1 cells, transiently transfected with human prostaglandin EP1 receptor