2.3. Single-Cell Calcium Imaging

These experiments were conducted with the Ca²⁺ probe Fluo4 as previously described [13,16,17]. Briefly, cells were loaded with 5 µM Fluo4/AM for 25 min, washed, and kept for another 5–10 min in a standard recording saline solution containing (in mM) 150 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 5.5 glucose, and 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4). All these procedures were performed at room temperature and in the dark. Coverslips were then mounted on the stage of an inverted Axio Observer A1 microscope with a Fluar 40× oil immersion objective lens (1.3 NA) (Carl Zeiss, Marly le Roi, France) and a charge-coupled device camera (CoolSnap HQ2, Princeton Instruments, Roper Scientific, Evry, France). The experimental setup, equipped with a DG-4 wavelength switcher (Princeton Instruments, Roper Scientific, France), was driven by MetaFluor (Universal Imaging, Roper Scientific, Evry, France). The cytosolic Ca²⁺ signals were acquired from cells bodies at a sampling rate of 0.2 Hz.