N a 1.30 plan apo objective

Cyclic AMP was assessed using FRET-based assays. Cells were transiently transfected with the FRET-based biosensors, Epac1-CFP/YFP^{54 (link)} for measuring cAMP and PTHR-CFP with βarr2-YFP for measuring arrestin recruitment. Measurements were performed and analyzed as previously described^{41 (link)}. In brief, cells plated on poly-D-lysine coated glass coverslips were mounted in Attofluor cell chambers (Life Technologies), maintained in Hepes buffer containing 150 mM NaCl, 20 mM Hepes, 2.5 mM KCl and 0.1–10 mM CaCl₂, 0.1% BSA, pH 7.4, and transferred on the Nikon Ti-E equipped with an oil immersion 40X N.A 1.30 Plan Apo objective and a moving stage (Nikon Corporation). CFP and YFP were excited using a mercury lamp. Fluorescence emissions were filtered using a 480 ± 20 nm (CFP) and 535 ± 15 nm (YFP) filter set and collected simultaneously with a LUCAS EMCCD camera (Andor Technology) using a DualView 2 (Photometrics) with a beam splitter dichroic long pass of 505 nm. Fluorescence data were extracted from single cell using Nikon Element Software (Nikon Corporation). The FRET ratio for single cells was calculated and corrected as previously described^{41 (link)}. Individual cells were perfused with buffer or with the ligand for the time indicated by the horizontal bar.