Fura-2 Calcium Imaging of hiPSC-Islet Grafts

Fura-2 loading was performed in retrieved intraocular hiPSC-islet grafts. The grafts were incubated with 2 µM fura-2 LeakRes/AM for 60 min at 37 °C in HEPES-buffered solution containing 125 mM NaCl, 5.9 mM KCl, 2.56 mM CaCl₂, 1.2 mM MgCl₂, 25 mM HEPES, 2 mM glucose and 0.1% bovine serum albumin (pH 7.4). Thereafter, fura-2-loaded hiPSC-islet grafts were placed and immobilized onto a glass coverslip at the bottom of a recording chamber. [Ca²⁺]_i was measured using a Spex Fluorolog spectrophotometer coupled to a Zeiss Axiovert 35 M microscope with a Zeiss Fluar 40×/l.30 oil objective (Carl Zeiss, Göttingen, Germany). The fura-2 F340/F380 ratio was registered to denote [Ca²⁺]_i [33 (link)]. During a recording, a hiPSC-islet graft was continuously perifused with HEPES-buffered solution supplemented with 2 mM glucose, 20 mM glucose or 30 mM KCl at 37 °C. Evtra software was employed to analyze the obtained data [27 (link)].