Measuring insulin concentrations

Insulin secretion into the culture medium from MIN6 cells was measured using the Mouse Insulin H-type^TM Enzyme-Linked Immunosorbent Assay Kit (Shibayagi Co., Ltd.), according to the manufacturer’s protocol. Briefly, MIN6 cells were cultured for 1 − 2 h in Krebs–Ringer bicarbonate (KRB) buffer (129 mM NaCl, 3.6 mM KCl, 0.5 mM NaH₂PO₄, 0.5 mM MgSO₄, 1.5 mM CaCl₂, 2 mM NaHCO₃, 10 mM HEPES, and 0.1% bovine serum albumin) containing 2.0 mM glucose. Next, the KRB buffer was changed to a buffer containing the indicated concentration of glucose (2.0 mM or 20 mM), and the cells were cultured for 1 h. The KRB buffer was collected and the insulin concentration was measured.