2.5. [Ca2+]c measurements

Both isolated islets and dispersed islet cells were perifused at 37 °C at the flow rate of 0.5 ml/min. A Nikon Eclipse TE2000-E inverted microscope equipped with a confocal QLC100 spinning disk (Yokogawa) was used to measure changes in GCaMP6f fluorescence in α-cells within whole isolated islets. This system allows [Ca²⁺]_c measurements in each α-cell of the islet instead of a global fluorescence of the whole islet. For dispersed cells, a confocal system was not needed, and a Zeiss Axiovert 100 inverted microscope was used instead. For confocal imaging, the GCaMP6f probe was excited at 491 nm and emission fluorescence was recorded at 503–552 nm. For epifluorescence imaging, the GCaMP6f probe was excited at 485 nm and the emitted fluorescence was recorded at 510–560 nm. Images were acquired every 1–3 s with an EMCCD QuantEM 512SC or a sCMOS Prime 95B camera (Photometrics) using Metafluor software (Molecular Devices).