GloSensor cAMP Assay

Human lung fibroblasts were electroporated with the GloSensor cAMP plasmid (Promega) using the 4D-Nucleofector System and P2 primary cell 4D-Nucelofector X Kit S (Lonza). Cells (10⁶) were suspended in Nucleofector Solution containing plasmid DNA (4 μg) before transfer to a Nucleocuvette. Electroporation was performed using programme EN150. Following electroporation, cells were resuspended in 500 μL complete culture media and seeded into a 384-well plate (20,000 cells/well) for 24 h prior to assay.

The GloSensor™ assay was carried out according to the manufacturer’s instructions (Promega). Briefly, media was aspirated, and cells were incubated in CO₂-independent media containing 4% v/v GloSensor™ cAMP reagent and incubated for 2 h at final experimental temperature of 37°C. Luminescence was measured on a BMG LABTEK ClarioStar plate reader, with 1 read per well every 3 min, over a period of 30 min at time: −0.5 h (prior to addition of ligand, 0 time point), 0 h (30 min time point), 2.5 h (3 h time point) or 23.5 h (24 h time point). Fibroblasts were stimulated with vehicle control (0.1% v/v DMSO), IPR ligands at a supra-maximal concentration (3 μM), or 10 μM forskolin (positive control). Data were analysed by calculating the area under the curve (AUC) over the 30 min measurement period. The AUC is expressed relative to the forskolin response at the same time point. Data are shown as the mean ± standard error of the mean of n biological replicates, as stated.