Insulin secretion in MIN6 cells following glucose stimulation.

MIN6 cells were cultured at a density of 3.0 × 10⁵ cells on a 12-well plate for 48 hours after electroporation (MPK10025, Thermo Fisher Scientific) as described above. The cells were preincubated with a 2.8 mmol/L (low glucose), Krebs-Ringer Modified Buffer (KRB) containing 10 mM HEPES (pH 7.4), 110 mM NaCl, 4.4 mM KCl, 1.45 mM KH₂PO₄, 1.2 mM MgSO₄, 2.3 mM calcium gluconate, 4.8 mM NaHCO₃, 4 mM glucose, and 0.3% bovine serum albumin for 30 minutes, and stimulated with 16.7 mmol/L (high glucose) glucose and 30 mmol/L KCL for 30 minutes. Insulin levels in the supernatant (release) and acid ethanol extraction (content) were measured using an Insulin Ultra-sensitive Assay kit (62IN2PEG, CisBio). The amount of secreted insulin was calculated as a ratio of release to content.