cAMP Accumulation Assay

cAMP accumulation was measured in duplicate using the LANCE cAMP accumulation assay (PerkinElmer). Human lung fibroblasts were seeded into 96-well plates and grown to confluency. On the day of the experiment, cells were incubated with vehicle control (0.1% v/v DMSO), IPR ligands, or a positive control (100 μM forskolin) diluted in stimulation buffer [Hanks’ balanced salt solution (HBSS) with 5 mM HEPES, 5.6 mM glucose, 1.3 mM CaCl₂, 0.1% (w/v) HSA (pH 7.4) and 5 μM rolipram] for 2 h at 37°C. For experiments using the IPR antagonist, RO-1138452, cells were pre-incubated with increasing concentrations of the antagonist for 10 min prior to addition of an EC₈₀ concentration of treprostinil (630 nM). To terminate the reaction, the buffer was aspirated, and 50 μL of ice-cold ethanol was added per well and left to evaporate at room temperature overnight. The cell precipitate was resuspended in 75 μL of lysis buffer [5 mM HEPES, 0.3% Tween 20, and 0.1% (w/v) BSA (pH 7.4)], and then, 5 μL was transferred to a 384-well white OptiPlate (PerkinElmer) on ice. After addition of AlexaFluor647-anti-cAMP antibody and Europium-labelled streptavidin with biotinylated cAMP for 2 h, the plate was read using an EnVision Multilabel Plate Reader (PerkinElmer) using LANCE settings. The data were analyzed against a cAMP standard curve using GraphPad Prism. To account for the inter-assay variation in levels of cAMP produced in each experiment, data were normalised to the forskolin response.