Assessment of agonist activity at recombinant EP2 and EP4 receptors (cAMP assay)

HEK cells stably expressing human EP₂ or EP₄ receptors (Wang et al., 2016) were routinely cultured at 37°C in a humidified 5% CO₂ incubator. The culture medium used was DMEM supplemented with 10% FBS, 100 U·mL⁻¹ penicillin, 100 ng·mL⁻¹ streptomycin, 2.5 μg·mL⁻¹ fungizone, 2 mM glutamine and 250 μg·mL⁻¹ geneticin. In addition, 200 μg·mL⁻¹ zeocin was included in the EP₂ cell media, while 200 μg·mL⁻¹ hygromycin was included in the EP₄ cell media.

For assay, cells were prepared at a density of 50 000 cells per well in 96‐well poly‐l‐lysine‐coated plates and allowed to grow to confluence (3–4 days) prior to use. Culture media were rinsed off using DMEM and replaced with DMEM containing the PDE inhibitor IBMX (1 mM) and the cycloxygenase inhibitor indomethacin (3 μM). This was allowed to incubate for 1 h before the cells were stimulated with PGE₂ or PGN compounds (in duplicate) for 15 min at final concentrations ranging from 0.0001 to 10 μM. The assay was terminated by the addition of 25 μL hydrochloric acid (1 N). Plates were then frozen for a minimum of 12 h or until required for radioligand displacement assay.

The cAMP radioligand displacement assay was as follows. Plates were thawed quickly at 37°C, and neutralized with 25 μL sodium hydroxide (1 N). Samples of supernatant (30 μL) were transferred to 96‐well Millipore Multiscreen_HTS‐FB (1 μm) plates coated with 0.1% polyethylenimine. These samples were diluted by addition of 90 μL cAMP assay buffer (50 mM Tris, 5 mM EDTA, pH 7.0). A cAMP standard curve (from 10⁻¹¹ to 10⁻⁵ M) was constructed. A 15 μL of 3′:5′‐cAMP‐dependent protein kinase (final concentration 8 μg per well) and 15 μL [³H]‐cAMP (final concentration 2 nM per well) were added to each well. Plates were incubated on ice for 2 h, before bound and free radiolabels were separated by vacuum filtration harvesting on a Millipore manifold, using ice‐cold water as the termination buffer. Filter plates were allowed to dry overnight, before addition of 50 μL Microscint 0. Radioactivity was determined using the Microbeta Trilux scintillation counter. cAMP accumulation was determined from the standard curve.