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HyPer7 and mCherry were imaged for 3 min baseline in imaging buffer, followed by two washes and replacement of media with prewarmed intracellular buffer (in mM): 10 NaCl, 130 KCl, 1 MgCl 2 , 1 KH 2 PO 4 , 2 succinic acid, and 20 HEPES (pH 7.4) supplemented with 0.5 mg/ml trypan blue and 20 M digitonin