Fluorescence Imaging of Intracellular Ca2+ Dynamics

Cells grown in 6-well plates were directly visualized with a DMi8 widefield microscope (Leica Microsystems). Phase-contrast and fluorescent images were acquired with a DFC9000 GT sCMOS camera using LAS X Premium software (Leica Microsystems). For Ca²⁺ imaging, cells were grown in 35 mm µ-dishes (ibidi) until the desired stage. Before imaging, cells were rinsed once with recording medium (128 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 45 mM sucrose, 10 mM glucose and 10 mM HEPES; pH 7.4) and incubated in this medium with 4 µg/mL of Fluo-8-AM for 30 min at 37 °C, as described by Carola et al. [31 (link)]. Cells were washed once with recording medium to remove residual Fluo-8, replenished with fresh recording medium and immediately imaged on a DMi8 widefield microscope equipped with an on-stage incubator (Pecon/Leica Microsystems).