Simultaneous optical measurements of cytosolic Ca2+ and cAMP in single cells

Understanding the temporal and spatial integration of the Ca2+ and adenosine 3',5'-monophosphate (cAMP) signaling pathways requires concurrent measurements of both second messengers. Here, we describe an optical technique to simultaneously image cAMP and Ca2+ concentration gradients in MIN6 mouse insulinoma cells using Epac1-camps, a Förster (or fluorescence) resonance energy transfer (FRET)-based cAMP biosensor, and Fura-2, a fluorescent indicator of Ca2+. This real-time imaging method allows investigation of the dynamic organization and integration of multiple levels of signal processing in single living cells.