Measuring cAMP in HEK-293T cells

Signaling experiments have been performed as previously described (Navarro et al., 2010 (link), 2016 (link), 2018b (link); Hinz et al., 2018 (link)). Two hours before initiating the experiment, HEK-293T cell-culture medium was replaced by serum-starved DMEM medium. Then, cells were detached, resuspended in growing medium containing 50 μM zardaverine and placed in 384-well microplates (2,500 cells/well). Cells were pretreated (15 min) with CBG -or vehicle- and stimulated with agonists (15 min) before adding 0.5 μM forskolin or vehicle. Readings were performed after 15 min incubation at 25°C. HTRF energy transfer measures were performed using the Lance Ultra cAMP kit (PerkinElmer, Waltham, MA, United States). Fluorescence at 665 nm was analyzed in a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMG Lab Technologies, Offenburg, Germany).