Glucose-stimulated insulin release (GSIS) in MIN6 cells

GSIS from MIN6 cells was determined using a static incubation protocol as in [22] and [12]. MIN6 cells were cultured in 96-well plates until ∼80% confluency, and the medium (with 25 mM glucose) was changed every 48 h. On the day of the experiment, growth medium was removed, and the cells were washed twice with glucose-free HEPES-balanced Krebs-Ringer phosphate buffer (KRBH; 111 mM NaCl, 25 mM NaHCO₃ (pH 7.4), 4.8 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 10 mM HEPES, 2.3 mM CaCl₂ and 0.1% BSA). Cells were preincubated for 1 h in 5% CO₂ at 37°C in KRBH supplemented with 1 mM glucose. The pre-incubation medium was removed, and the cells were washed once in glucose-free KRBH. The cells were then incubated for 1 h in 20 mM glucose-containing KRBH in the absence (control, CTL) or presence of BF142. At the same time, a set of control cells were incubated for 1 h in 1 mM glucose-containing KRBH for determination of insulin baseline. For all experiments, incubation medium was collected, spun at 1500 g for 5 min 4°C, and then diluted 20X. Insulin concentration in diluted samples was determined using a Mouse Insulin ELISA Kit (Mercodia, Winston Salem, N.C., USA). After the assay, 1 M NaOH was added to the adherent MIN6 cells and the total protein content was determined using BCA reagent (Pierce^TM BCA Protein Assay Kit, Thermofisher, Walthman, MA, USA). Insulin secretion was normalized to protein content.