Measurement of Intracellular Calcium

Intracellular calcium was measured by imaging fluo-4 AM. Cells cultured on glass-bottomed tissue culture dishes (Corning incorporated, Corning, NY, United States) were rinsed by loading buffer (150 μM NaCl, 5 mM KCl, 1 μM MgCl, 10 μM glucose, and 20 μM HEPES, pH 7.4) twice and then bathed with fluo 4-AM solution (2.5 μM, Invitrogen Corp., Carlsbad, CA, United States), which is dissolved in loading buffer supplemented with 2% BSA and 0.01 % Pluronic F-127 (BASF) protected for light for 25 min at room temperature on a rolling plate. Cells were then washed twice using loading buffer and incubated in serum-free DMEM at 30°C for 20 min to allow the de-esterification.

Cells were cultured on glass-bottomed tissue culture dishes. Cells were rinsed twice with loading buffer followed by a 25-min incubation with 20 μM BAPTA-AM (Invitrogen, Carlsbad, CA, United States) dissolved in loading buffer supplemented with 2% BSA and 0.01 % Pluronic F-127 (BASF, Sigma, St Louis, MO, United States) on a rolling plate agitator at room temperature. After loading, cells were then washed twice with loading buffer and incubated in serum-free DMEM for 20 min at 30°C to allow the de-esterification.