Figure 5

The isolated pancreatic islets were incubated for 4 hours at room temperature in 1 ml of RPMI medium containing 5 μM of Ca²⁺ fluorescent dye, fluo 4-AM. The images were continuously acquired before and after addition of 10^-7 M of glutamate, at time intervals of 1.318 seconds, for a total of 600 seconds. Time series experiments were performed collecting 512 × 512 pixel images at 400 Hz. Fluorescence intensity was analysed using the quantitation mode in LAS-AF software from Leica Microsystems, Germany. A region of interest (ROI) was drawn within a field of view. The pixel intensity was calculated for each image in the 600 seconds sequence to analyse the intracellular Ca²⁺ release from the pancreatic islet cells in experimental conditions.