Cells were plated on 25-mm optical borosilicate poly-l-lysine–coated sterile cover glasses (Sigma) at 80% confluence and loaded with 1 μM Mag-Fura-2 AM in Ringer solution for 60 min at 37°C. For permeabilization, cells were perfused (5 ml/min) with 2 mM Ca2+ Ringer solution for 1 min followed by perfusion with 0.01 saponin for 1 min in intracellular-like medium containing 100 nM Ca2+ and 5 μM of the reversible SERCA inhibitor tBHQ. The solution was then switched to intracellular-like medium without saponin for 3 min to allow ER Ca2+ refilling. The intracellular-like medium contained 130 mM KCl, 1 mM KH2PO4, 1 mM MgCl2, 1 mM ATP, 5 mM Na-succinate, 5 mM Na-pyruvate, 20 mM Na-Hepes (pH 7.0), and Ca2+ buffered at 100 nM (with titrated 0.6 mM EGTA and EGTA-Ca2+ solutions). Fluorescence intensities were recorded as indicated above. For GSH inhibition, cells were pretreated with 2 mM BSO for 2 hours before cell permeabilization.

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