Insulin secretion in vitro

Single islets of similar size were transferred to a 96-well plate and were subsequently incubated at 37 °C on a shaking platform (300 r.p.m.) for 30 min each in solutions containing different stimuli. Afterwards islets were lysed and released insulin was normalized to the total islet insulin content. Standard secretion buffer contained (in mM) 98 NaCl, 0.9 CaCl₂, 2.7 KCl, 1.5 KH₂PO₄, 0.5 MgCl₂, 8 Na₂HPO₄, 20 HEPES, 0.2% BSA, pH 7.4 with NaOH. Different concentrations of glucose (3.3, 10 or 25 mM) or 300 µM tolbutamide were added and the osmolarity of the solutions was balanced by adding mannitol (290 mOsm). Islets that showed morphological changes indicative of lysis or cell death at the end of the secretion period were excluded from the analysis. Islets were lysed afterwards to normalize secreted insulin amounts to the total insulin content. Insulin levels in the supernatant or from total pancreatic lysates were measured using an ultrasensitive insulin ELISA (Alpco, 80-INSMSU-E01) according to manufacturer’s instructions. The genotype of islets was revealed only after the end of the experiment.