Intracellular Ca2+ Imaging in Pancreatic Islets

Intracellular free Ca²⁺ was imaged in KHB saturated with 95% O₂/5% CO₂ and adjusted to pH 7.4, with additions as stated in the figures. Islets were incubated at 37°C for 45 minutes in KHB containing 10 μM Fluo-2-AM (Cambridge Biosciences). Islets were then transferred in a perfusion chamber, mounted on a Zeiss Axiovert confocal microscope, and continuously perfused at 34°C–36°C. Fluo-2 was excited with a 491-nm laser line and emitted light filtered at 525/50 nm. Images were acquired with a Hamamatsu ImagEM camera, and Volocity software (PerkinElmer) was used for data capture. Traces are presented as normalized intensity over time (F/F₀) (49 (link)).