Measurement of Intracellular cAMP

All cAMP assays were performed in serum-free DMEM (Sigma) supplemented with 3-isobutyl-1-methylxanthine (IBMX; 0.5 mM; Sigma) to inhibit cAMP degradation by phosphodiesterases. cAMP concentrations were measured from cell lysates after cells were incubated for 5 min with synthetic agonists (10 μM) for FFAR2 (4-CMTB) or FFAR3 ({"type":"entrez-nucleotide","attrs":{"text":"AR420626","term_id":"40175736","term_text":"AR420626"}}AR420626) using the HTRF cAMP Dynamic 2 immunoassay kit (CisBio). Fluorescence was measured with a PHERAstar FSX plate reader (BMG Labtech) equipped with HTRF 337 optic module, with excitation at 340 nm and measurements of emission at 620 and 665 nm. cAMP levels were interpolated from an cAMP standard curve and normalized to protein concentration. All experiments were conducted in triplicate and repeated at least 3 times.