Monitoring Cytosolic Ca2+ Signaling in HEK293 Cells

Cytosolic Ca²⁺ levels in stable, inducible HEK293 cells expressing RyR2 WT or the H29D mutant were monitored using single-cell Ca²⁺ imaging and the fluorescent Ca²⁺ indicator dye Fura-2 as described previously [18 (link),26 (link),31 (link)]. Briefly, cells grown on glass coverslips for 8-18h after induction (as indicated) by 1 μg/ml tetracycline (Sigma) were loaded with 5 μM Fura-2, AM in KRH buffer (125 mM NaCl, 5 mM KCl, 6 mM glucose, 1.2 mM MgCl₂ and 25 mM Hepes, pH 7.4) plus 0.02% pluronic F-127 and 0.1 mg/ml BSA for 20 min at room temperature (23°C). The coverslips were then mounted in a perfusion chamber (Warner Instruments) on an inverted microscope (Nikon TE2000-S). The cells were perfused continuously with KRH buffer containing increasing extracellular Ca²⁺ concentrations (0, 0.1, 0.2, 0.3, 0.5, 1.0 and 2.0 mM). Caffeine (10 mM) was applied at the end of each experiment to confirm the expression of active RyR2 channels. Time-lapse images (0.25 frame/s) were captured and analyzed with Compix Simple PCI 6 software. Fluorescence intensities were measured from regions of interest centered on individual cells. Only cells that responded to caffeine were analyzed. The filters used for Fura-2 imaging were λex = 340±26 nm and 387±11 nm, and λem = 510±84 nm with a dichroic mirror (410 nM).