cAMP GloSensor assay

The GloSensor™ cAMP Assay (Promega) was used for detecting changes in the intracellular levels of cAMP. Initially, a Greiner Bio-One 96 Well Plate (white, TC treated) was seeded at 8000 cells per 100 μl well, as described above. After 24 h, the cells were transfected with the pGloSensor™ cAMP Plasmid using the Lipofectamine3000 transfection reagent as above. Between 24 and 48 h later, allowing for the accumulation of the biosensor, the medium in the 96-well plate was changed to CO₂-independent medium supplemented with GlutaMAX™ (Life Technologies), 10% foetal bovine serum and 2% GloSensor™ cAMP Reagent. The plate was then stored for 2 h at room temperature to equilibrate. Following the incubation period, the cells were treated with relevant drugs and their luminescence values were measured after 20 min in the BMG FLUOstar OPTIMA Microplate Reader, with forskolin as a positive control, and DMSO as a negative control.