Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at recombinant human EP3 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 EP3 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 EP3 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 EP3 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 EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at recombinant human EP3 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 EP3 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 EP3 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 EP3 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 EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at recombinant human EP3 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 EP3 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 EP3 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 EP3 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 EP3 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 EP3 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 EP3 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 EP3 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

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 EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM 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 recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assayAgonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assay

Agonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assayAgonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assayAgonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assay

Agonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assayAgonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 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 recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.

FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assayAgonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assay

Agonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assayAgonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assay

Agonist activity at recombinant human EP3 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 EP3 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 EP3 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 EP3 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 EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.

FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.

FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assayAgonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assay

Agonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assayAgonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assayAgonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assay

Agonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assayAgonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assayAgonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assay

Agonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assayAgonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at recombinant human EP3 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 EP3 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 EP3 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 EP3 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

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP3 (% of control ligand, sulprostone=130%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP3 (% of control ligand, sulprostone=130%)

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP3 (% of control ligand, sulprostone=130%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP3 (% of control ligand, sulprostone=130%)

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A 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 EP3 (% of control ligand, sulprostone=50%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP3 (% of control ligand, sulprostone=50%)

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP3 (% of control ligand, sulprostone=50%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP3 (% of control ligand, sulprostone=50%)

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP3 (% of control ligand, sulprostone=50%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP3 (% of control ligand, sulprostone=50%)

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP3 (% of control ligand, sulprostone=50%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP3 (% of control ligand, sulprostone=50%)

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP3 (% of control ligand, sulprostone=50%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP3 (% of control ligand, sulprostone=50%)

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assayAgonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assay

Agonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assayAgonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Antagonist activity at human EP3-2 receptor expressed in CHOK1 cellsAntagonist activity at human EP3-2 receptor expressed in CHOK1 cells

Antagonist activity at human EP3-2 receptor expressed in CHOK1 cellsAntagonist activity at human EP3-2 receptor expressed in CHOK1 cells

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP3 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay

Agonist activity at human EP3 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP3 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at recombinant human EP3 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 EP3 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 EP3 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 EP3 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 EP3 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 EP3 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 EP3 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 EP3 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 EP3 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 EP3 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 EP3 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 EP3 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 EP3 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 EP3 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 EP3 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 EP3 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 EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at recombinant human EP3 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 EP3 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 EP3 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 EP3 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 EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at recombinant human EP3 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 EP3 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 EP3 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 EP3 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 EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.

FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.

FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM 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 EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP3(% of control ligand, sulprostone=75%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP3(% of control ligand, sulprostone=75%)

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP3(% of control ligand, sulprostone=75%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP3(% of control ligand, sulprostone=75%)

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP3(% of control ligand, sulprostone=75%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP3(% of control ligand, sulprostone=75%)

Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP3(% of control ligand, sulprostone=75%)Functional activity in RAT-1cells, transiently-transfected with human Prostaglandin E receptor EP3(% of control ligand, sulprostone=75%)

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.

FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.

FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assayAgonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assay

Agonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assayAgonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assay

Agonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assayAgonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assay

Agonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assayAgonist activity at recombinant human EP3A receptor expressed in HEK293-EBNA cells co-expressing Gqi5 assessed as induction of calcium mobilization after 45 mins by fluo-4AM dye-based FLIPR assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysisAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by fluorescence based analysis

Agonist activity at recombinant human EP3 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 EP3 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 EP3 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 EP3 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

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 EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assayAgonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay

FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.

FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.

FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.

FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.FLIPR Assay: Calciium Signal Studies of the Flipr. Cells were seeded at a density of 5×104 cells per well in Biocoati® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 μM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 μl in each well. Plates were re-equilibrated to 37° C. for a few minutes. Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration.

Antagonist activity at human EP3 receptor assessed as cAMP production by cell-based assayAntagonist activity at human EP3 receptor assessed as cAMP production by cell-based assay

Antagonist activity at human EP3 receptor assessed as cAMP production by cell-based assayAntagonist activity at human EP3 receptor assessed as cAMP production by cell-based assay

Antagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albuminAntagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albumin

Antagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albuminAntagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albumin

Antagonist activity at human EP3 receptor assessed as cAMP production by cell-based assayAntagonist activity at human EP3 receptor assessed as cAMP production by cell-based assay

Antagonist activity at human EP3 receptor assessed as cAMP production by cell-based assayAntagonist activity at human EP3 receptor assessed as cAMP production by cell-based assay

Antagonist activity at human EP3 receptor expressed in CHO-K1 cells assessed as reduction in sulprostone induced inhibition of forskolin stimulated cAMP production incubated for 30 mins by TR-FRET assayAntagonist activity at human EP3 receptor expressed in CHO-K1 cells assessed as reduction in sulprostone induced inhibition of forskolin stimulated cAMP production incubated for 30 mins by TR-FRET assay

Antagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albuminAntagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albumin

Antagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albuminAntagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albumin

Antagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albuminAntagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albumin

Antagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albuminAntagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albumin

Antagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assayAntagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assay

Antagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assayAntagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assay

Antagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by fluorescence assay in presence of 1% bovine serum albuminAntagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by fluorescence assay in presence of 1% bovine serum albumin

Antagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by fluorescence assay in presence of 1% bovine serum albuminAntagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by fluorescence assay in presence of 1% bovine serum albumin

Antagonist activity at human EP3 receptor expressed in CHO-K1 cells assessed as reversal of inhibition of forskolin-induced cAMP productionAntagonist activity at human EP3 receptor expressed in CHO-K1 cells assessed as reversal of inhibition of forskolin-induced cAMP production

Antagonist activity at human EP3 receptor expressed in CHO-K1 cells assessed as reversal of inhibition of forskolin-induced cAMP productionAntagonist activity at human EP3 receptor expressed in CHO-K1 cells assessed as reversal of inhibition of forskolin-induced cAMP production

Antagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by fluorescence assay in presence of 1% bovine serum albuminAntagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by fluorescence assay in presence of 1% bovine serum albumin

Antagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by fluorescence assay in presence of 1% bovine serum albuminAntagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by fluorescence assay in presence of 1% bovine serum albumin

Antagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assayAntagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assay

Antagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assayAntagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assay

Antagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by fluorescence assay in presence of 1% bovine serum albuminAntagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by fluorescence assay in presence of 1% bovine serum albumin

Antagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by fluorescence assay in presence of 1% bovine serum albuminAntagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by fluorescence assay in presence of 1% bovine serum albumin

Antagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by fluorescence assay in presence of 1% bovine serum albuminAntagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by fluorescence assay in presence of 1% bovine serum albumin

Antagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by fluorescence assay in presence of 1% bovine serum albuminAntagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by fluorescence assay in presence of 1% bovine serum albumin

Antagonist activity at human EP3 expressed in CHO cells assessed as suppression of sulprostone-induced inhibition of forskolin induced cAMP production measured after 30 mins by HTR-FRET assayAntagonist activity at human EP3 expressed in CHO cells assessed as suppression of sulprostone-induced inhibition of forskolin induced cAMP production measured after 30 mins by HTR-FRET assay

Antagonist activity at human EP3 expressed in CHO cells assessed as suppression of sulprostone-induced inhibition of forskolin induced cAMP production measured after 30 mins by HTR-FRET assayAntagonist activity at human EP3 expressed in CHO cells assessed as suppression of sulprostone-induced inhibition of forskolin induced cAMP production measured after 30 mins by HTR-FRET assay

Antagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assayAntagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assay

Antagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assayAntagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assay

Antagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albuminAntagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albumin

Antagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albuminAntagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albumin

Antagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase intracellular calcium level in presence of 1% BSA by fluorimetryAntagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase intracellular calcium level in presence of 1% BSA by fluorimetry

Antagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase intracellular calcium level in presence of 1% BSA by fluorimetryAntagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase intracellular calcium level in presence of 1% BSA by fluorimetry

Antagonist activity at human EP3 expressed in CHO cells assessed as suppression of sulprostone-induced inhibition of forskolin induced cAMP production measured after 30 mins by HTR-FRET assayAntagonist activity at human EP3 expressed in CHO cells assessed as suppression of sulprostone-induced inhibition of forskolin induced cAMP production measured after 30 mins by HTR-FRET assay

Antagonist activity at human EP3 expressed in CHO cells assessed as suppression of sulprostone-induced inhibition of forskolin induced cAMP production measured after 30 mins by HTR-FRET assayAntagonist activity at human EP3 expressed in CHO cells assessed as suppression of sulprostone-induced inhibition of forskolin induced cAMP production measured after 30 mins by HTR-FRET assay

Antagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assayAntagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assay

Antagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assayAntagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assay

Antagonist activity at human EP3 receptor expressed in CHO-K1 cells assessed as reduction in sulprostone induced inhibition of forskolin stimulated cAMP production incubated for 30 mins by TR-FRET assayAntagonist activity at human EP3 receptor expressed in CHO-K1 cells assessed as reduction in sulprostone induced inhibition of forskolin stimulated cAMP production incubated for 30 mins by TR-FRET assay

Antagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase intracellular calcium level in presence of 1% BSA by fluorimetryAntagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase intracellular calcium level in presence of 1% BSA by fluorimetry

Antagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase intracellular calcium level in presence of 1% BSA by fluorimetryAntagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase intracellular calcium level in presence of 1% BSA by fluorimetry

Antagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by fluorescence assay in presence of 1% bovine serum albuminAntagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by fluorescence assay in presence of 1% bovine serum albumin

Antagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by fluorescence assay in presence of 1% bovine serum albuminAntagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by fluorescence assay in presence of 1% bovine serum albumin

Antagonist activity at human EP3 expressed in CHO cells assessed as suppression of sulprostone-induced inhibition of forskolin induced cAMP production measured after 30 mins by HTR-FRET assayAntagonist activity at human EP3 expressed in CHO cells assessed as suppression of sulprostone-induced inhibition of forskolin induced cAMP production measured after 30 mins by HTR-FRET assay

Antagonist activity at human EP3 expressed in CHO cells assessed as suppression of sulprostone-induced inhibition of forskolin induced cAMP production measured after 30 mins by HTR-FRET assayAntagonist activity at human EP3 expressed in CHO cells assessed as suppression of sulprostone-induced inhibition of forskolin induced cAMP production measured after 30 mins by HTR-FRET assay

Antagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albuminAntagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albumin

Antagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albuminAntagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albumin

Antagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level by fluorescence assay in presence of 1% BSAAntagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level by fluorescence assay in presence of 1% BSA

Antagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level by fluorescence assay in presence of 1% BSAAntagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level by fluorescence assay in presence of 1% BSA

Antagonist activity at human EP3 receptor expressed in CHO-K1 cells assessed as reduction in sulprostone induced inhibition of forskolin stimulated cAMP production incubated for 30 mins by TR-FRET assayAntagonist activity at human EP3 receptor expressed in CHO-K1 cells assessed as reduction in sulprostone induced inhibition of forskolin stimulated cAMP production incubated for 30 mins by TR-FRET assay

Antagonist activity at human EP3 receptor expressed in CHO-K1 cells assessed as inhibition of forskolin-induced cAMP level after 10 mins by EIAAntagonist activity at human EP3 receptor expressed in CHO-K1 cells assessed as inhibition of forskolin-induced cAMP level after 10 mins by EIA

Antagonist activity at human EP3 receptor expressed in CHO-K1 cells assessed as inhibition of forskolin-induced cAMP level after 10 mins by EIAAntagonist activity at human EP3 receptor expressed in CHO-K1 cells assessed as inhibition of forskolin-induced cAMP level after 10 mins by EIA

Antagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assayAntagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assay

Antagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assayAntagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assay

Antagonist activity at human EP3 receptor expressed in CHO-K1 cells assessed as reduction in sulprostone induced inhibition of forskolin stimulated cAMP production incubated for 30 mins by TR-FRET assayAntagonist activity at human EP3 receptor expressed in CHO-K1 cells assessed as reduction in sulprostone induced inhibition of forskolin stimulated cAMP production incubated for 30 mins by TR-FRET assay

Antagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assayAntagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assay

Antagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assayAntagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assay

Antagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assayAntagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assay

Antagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assayAntagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assay

Antagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level by fluorescence assay in presence of 0.1% BSAAntagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level by fluorescence assay in presence of 0.1% BSA

Antagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level by fluorescence assay in presence of 0.1% BSAAntagonist activity at mouse EP3alpha receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level by fluorescence assay in presence of 0.1% BSA

Antagonist activity at mouse EP3alpha receptor expressed in CHo cells assessed as inhibition of PGE2-stimulated intracellular calcium concentrationAntagonist activity at mouse EP3alpha receptor expressed in CHo cells assessed as inhibition of PGE2-stimulated intracellular calcium concentration

Antagonist activity at mouse EP3alpha receptor expressed in CHo cells assessed as inhibition of PGE2-stimulated intracellular calcium concentrationAntagonist activity at mouse EP3alpha receptor expressed in CHo cells assessed as inhibition of PGE2-stimulated intracellular calcium concentration

Antagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assayAntagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assay

Antagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assayAntagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assay

Antagonist activity at human EP3 receptor expressed in CHO-K1 cells assessed as reduction in sulprostone induced inhibition of forskolin stimulated cAMP production incubated for 30 mins by TR-FRET assayAntagonist activity at human EP3 receptor expressed in CHO-K1 cells assessed as reduction in sulprostone induced inhibition of forskolin stimulated cAMP production incubated for 30 mins by TR-FRET assay

Antagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase intracellular calcium level in presence of 1% BSA by fluorimetryAntagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase intracellular calcium level in presence of 1% BSA by fluorimetry

Antagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase intracellular calcium level in presence of 1% BSA by fluorimetryAntagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase intracellular calcium level in presence of 1% BSA by fluorimetry

Antagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase intracellular calcium level in presence of 1% BSA by fluorimetryAntagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase intracellular calcium level in presence of 1% BSA by fluorimetry

Antagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase intracellular calcium level in presence of 1% BSA by fluorimetryAntagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase intracellular calcium level in presence of 1% BSA by fluorimetry

Antagonist activity at human EP3 expressed in CHO cells assessed as suppression of sulprostone-induced inhibition of forskolin induced cAMP production measured after 30 mins by HTR-FRET assayAntagonist activity at human EP3 expressed in CHO cells assessed as suppression of sulprostone-induced inhibition of forskolin induced cAMP production measured after 30 mins by HTR-FRET assay

Antagonist activity at human EP3 expressed in CHO cells assessed as suppression of sulprostone-induced inhibition of forskolin induced cAMP production measured after 30 mins by HTR-FRET assayAntagonist activity at human EP3 expressed in CHO cells assessed as suppression of sulprostone-induced inhibition of forskolin induced cAMP production measured after 30 mins by HTR-FRET assay

Antagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assayAntagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assay

Antagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assayAntagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assay

Antagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assayAntagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assay

Antagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assayAntagonist activity at human EP3 receptor expressed in CHO cells by cell based cAMP functional assay

Antagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albuminAntagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albumin

Antagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albuminAntagonist activity at mouse EP3 receptor expressed in CHO cells assessed as inhibition of PGE2-induced increase in intracellular calcium level after 1 hr by FDSS in presence of 1% bovine serum albumin