BRET Assay for cAMP Signaling

Transiently transfected cells were washed and preincubated at 37 °C and 5% CO₂ for 20 min in 40 µL of phosphate buffered saline (PBS) added with 1 mM HEPES and 500 µM of the phosphodiesterase inhibitor 3-isobutil-1-methylxanthine (IBMX). Then, cells were washed by PBS and maintained for 30 min at 37 °C and 5% CO₂ in 50 µL of PBS and 1 mM HEPES, in the presence or absence of increasing doses of LH or hCG (1 pM–100 nM range) [10 (link)]. Cells treated with 50 µM forskolin (Sigma-Aldrich) served as positive controls [31 (link)]. Bioluminescence resonance energy transfer (BRET) measurements were performed upon addition of 10 µL of 5 µM coelenterazine H (Interchim, Montluçon, France). Signals emitted by donor and acceptor tags were detected by the CLARIOstar microplate reader (BMG Labtech, Ortenberg, Germany) at wavelengths of 480 ± 20 and 540 ± 20 nm, respectively, and represented as a ratio (BRET changes) [32 (link)].