Intracellular cAMP Dynamics Measurement by FRET

To evaluate intracellular cAMP levels, fluorescence resonance energy transfer (FRET) experiments were performed. Briefly, HCT8 cells were seeded onto 12 mm diameter glass coverslips at 37°C, 5% CO_2, and transiently transfected with a plasmid encoding the H96 sensor (Klarenbeek et al., 2011 (link)) (a gift from Dr. K. Jalink) containing the cAMP-binding sequence of Epac1 sandwiched between ECFP (donor) and EYFP (acceptor). In detail, cells were transfected with 0.5 μg of DNA/cm² using TransFectin™ Lipid Reagent (1.5 μL/cm²) according to the protocol provided by the manufacturer (Bio-Rad Laboratories, Inc., Hercules, CA, United States) and left under basal condition or treated as mentioned before. Experiments were performed 48 h after transfection. Visualization of ECFP- and/or EYFP-expressing cells and detection of FRET was performed on an inverted microscope (Nikon Eclipse TE2000-S) controlled by Metamorph^® Microscopy Automation and Image Analysis Software (Molecular Devices, LLC, San Jose, CA, United States). Each image was corrected and analyzed as previously shown (Di Mise et al., 2019 (link)).