Measuring Cellular cAMP Levels

Two hours before initiating the experiment, HEK-293T or neuronal cell-culture medium was exchanged to serum-starved DMEM or neurobasal medium, as corresponds. Then, cells were detached, resuspended in the serum-starved medium containing 50 µM zardaverine, plated in 384-well microplates (2500 cells/well), pre-treated (15 min) with the corresponding antagonists or the vehicle, and then stimulated with agonists (15 min) before adding 0.5 μM forskolin or vehicle. Readings were performed after 1 h incubation at 25 °C. Homogeneous time-resolved fluorescence energy transfer (HTRF) measures were obtained using the Lance Ultra cAMP kit (PerkinElmer, Waltham, MA, USA) [62 (link)]. Fluorescence at 665 nm was analyzed on a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMG Lab technologies, Offenburg, Germany).