Islets were isolated as described above. Simultaneous imaging of Ca2+ of individual cells was performed by spinning disc confocal microscopy after loading intact islets with Fluo 2-AM (Cambridge Bioscience, Cambridge, UK). Images were captured with a Zeiss Axiovert 200M microscope fitted with a ×10 0.3–0.5 NA, EC Plan Neofluar, Zeiss objective and a ×1.5 Optivar attached to a Nokigawa spinning disc confocal head, as described (27). The microscope was controlled using Volocity software. Islets were continuously perifused in Krebs-Ringer buffer containing 3 mM glucose, equilibrated with 95% O2-5% CO2 at 34–36°C. Islets were stimulated at 210 s and 1,300 s by perifusion with Krebs-Ringer supplemented with up to 16.7 mM glucose or 20 mM KCl as indicated. Offline processing and analysis were conducted using ImageJ software (1) and an in-house macro as described under supplementary methods.