Individual tissue slice was transferred to an imaging chamber equipped with a perfusion system containing HB-ECS at 34°C and exposed to a square glucose stimulation pulse (8 mM) lasting for several tens of minutes. To study islet activation the pH of the HB-ECS was set to 7.4, 7.1 or 7.7 and islets were placed in a non-stimulatory glucose concentration (6 mM) at one of the pH levels, following stimulation with 8 mM glucose solution of the same pH (Figure 1). To study the pH-dependence of the plateau phase of the beta cell activity as well as the beta cell network properties, the pH of the extracellular solution was switched in the order 7.4 - 7.1 - 7.7 (sequence 1) or 7.4 - 7.7 - 7.1 (sequence 2) during the 1-hour exposure to stimulatory glucose of 8 mM (Figure 2). In the control experiments pH 7.4 was maintained throughout the duration of the experiment (Figures 2J–L). The imaging was performed on a Leica TCS SP5 upright confocal system (20x HCX APO L water immersion objective, NA 1.0). The acquisition frequency was set to 2 Hz and the resolution to 512x512. Calbryte 520 was excited by an argon 488 nm laser line and emitted light was detected by Leica HyD hybrid detector in the range of 500-700 nm (all from Leica Microsystems, Germany), as described previously (19 (link)).
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