Cell viability assay. Islet cell viability after 7 days of 16.7 mmol/l glucose culture was assessed using the Cell Proliferation Kit I according to the manufacturer’s instructions (Roche, Indianapolis, IN, USA). This assay is based on the ability of viable
MT2 to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to insoluble coloured formazan crystals. Briefly, islets were plated at 20/well (duplicates for each condition) and 0.5% (w/v) MTT was added to 100 μL of culture medium in each well. After 4 h at 37 °C, 100 μL solubilization solution was added. After overnight incubation, solubilized formazan was quantified spectrophotometrically. Perifusion of islets for insulin secretion. After 7 days of 16.7 mmol/l glucose culture, 100 islets were collected and loaded into a perifusion chamber. Islets were first perifused with Krebs–Ringer bicarbonate buffer (KRB; 98.5 mmol/l NaCl, 5.5 mmol/l NaHCO3, 4.9 mmol/l KCl, 1.2 mmol/l KH2PO4, 1.2 mmol/l MgSO4, 2.6 mmol/l CaCl2, 20 mmol/l Hepes, and 0.1% BSA, pH 7.4) containing 1.67 mmol/l
glucose for 1 h to achieve basal insulin secretion. Effluent fractions were then collected at 2–5 min intervals during perifusion with 1.67 mmol/l glucose for 8 min, then with 16.7 mmol/l glucose for 30 min, followed by 16.7 mmol/l glucose with 10 mmol/l arginine and 0.01 mmol/l IBMX for 30 min. Finally, islets were perifused with 1.67 mmol/l glucose for 20 min. Samples were stored at ?20 °C before determination of insulin by radioimmunoassay. Islet insulin content was also measured after acid-ethanol extraction.
