Real-Time Monitoring of Intracellular cAMP

For real-time monitoring of intracellular cAMP production, cells were transfected with Epac1-camps plasmid (1 μg/ 35 mm dish), kindly provided by M. Lohse (Germany), using X-tremeGENE transfection reagents (Roche applied science) as described previously (Jung et al. 2010 (link)). After 1–2 days, fluorescence was excited at 405 nm, and emission from CFP and YFP was detected from the whole cytoplasm at 420–480 nm (F_CFP) and 560–615 nm (F_YFP) using a confocal microscope (LSM 710; Carl Zeiss, Inc.). The F_CFP and F_YFP signals were corrected for background. To determine bleed-through corrections, we measured F_CFP and F_YFP for cells expressing CFP alone and YFP alone. 38% of the CFP signal appears in YFP channel and no YFP signals in CFP channel. Therefore, the final FRET ratio was calculated as (F_YFP-0.38*F_CFP)/F_CFP. A decrease of the FRET ratio represents an increase in intracellular cAMP concentration since cAMP binding to the Epac1 domain causes a conformational change for CFP-YFP separation.