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Before imaging, islets were loaded with 8 M Fluo-4 AM (Thermo Fisher) at room temperature (21C) for 90 minutes in imaging buffer consisting of 140 mM NaCl, 3.6 mM KCl, 0.5 mM MgSO 4 , 1.3 mM CaCl 2 , 10 mM HEPES (pH 7.4 with NaOH), 5 mM NaHCO 3 , and 0.5 mM NaH 2 PO 4 with either 5.5 mM or 16.7 mM glucose (reflecting the culture conditions), 1 mg/mL BSA, and 0.02% pluronic F-127 (Thermo Fisher)

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