Astrocytes grown on glass-bottom dishes were loaded with Fura-2 by incubating cells in 2 μM Fura-2–AM (Invitrogen) in growth medium for 35 min at 37°C. Fura-2–containing medium was washed off, and cells were incubated for an additional 5 to 10 min before imaging. All experiments were performed at room temperature. Single-cell [Ca2+]i measurements were performed as described previously (29). Image acquisition and analysis were performed using SlideBook (Denver, CO). Dishes were mounted on the stage of an Olympus IX71 inverted microscope, and images were acquired every 6 s at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. For data analysis, ROIs were drawn around single cells, background was subtracted, and F340/F380 ratios were calculated for each time point. A rise in the ratio of emission when excited at 340 nm over the ratio when excited at 380 nm indicated a rise in [Ca2+]i.

[Ca2+]i was estimated from F340/F380 ratio using the standard equation: [Ca2+]i = ßKd (RRmin)/(RmaxR), where R is the F340/F380 fluorescence ratio and values of Rmin and Rmax were determined from an in vitro calibration of Fura-2 pentapotassium salt. β was determined from the Fmin/Fmax ratio at 380 nm and Kd is the apparent dissociation constant of Fura-2 binding to Ca2+ (132 nM). The values of these parameters were as follows: Rmin = 0.36, Rmax = 19.37, ß = 12.18. For each cell, the rate of SOCE (∆[Ca2+]i/∆t) was calculated from the slope of a line fitted to three points (18 s) after the readdition of 2 mM Ca2+ Ringer’s solution. Baseline [Ca2+]i was calculated by averaging [Ca2+]i values over a 2-min baseline for each experiment. Store release was calculated by measuring the area under the curve during TG application in Ca2+-free solution.

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