Simultaneous Fluorescent Recordings of Extracellular ATP and IntracellularCalcium in Mammalian Cells

Extracellular ATP is a potent signaling molecule that stimulates intracellular calcium responses through purinergic (P2) receptors in mammalian cells. While extracellular ATP and intracellular calcium can be measured separately, simultaneous monitoring can offer additional insights into P2 receptor physiology. This protocol takes advantage of the overlapping fluorescence spectra between the ATP-detection substrate luciferin and calcium indicator dye Fura2. Mammalian cells are loaded with Fura2-AM and live-cell recordings are acquired in the presence of a luciferin-luciferase imaging solution. This protocol allows to study stimulus-induced ATP release and directly relate changes in extracellular ATP concentration to observed calcium responses.