MIN6 cells

A method to assay insulin secretion in vitro using MIN6 cells [53] has been described [54]. Briefly, MIN6 cells were maintained in Dulbecco’s modified Eagle’s medium containing 4.5 g/L D-glucose (Wako), 10% fetal bovine serum, 0.1 mM 2-mercaptoethanol, 100 units/ml penicillin, and 0.05 mg/ml streptomycin in humidified 5% CO₂ at 37°C. Cells were replated and cultured for 3 days, and then treated with 10 μg/ml LPS, 100 ng/ml recombinant soluble RANKL (R&D systems), or 100 ng/ml recombinant OPG (R&D systems) as indicated. After additional culture for 24 hr, cells were starved in HEPES-balanced Krebs-Ringer bicarbonate (HKRB) buffer with 3 mM glucose (Cosmobio) for 30 min and then incubated 1 hr in HKRB buffer containing 3, 9.8, or 20 mM D-glucose. Culture supernatants were collected for insulin measurement. Cell lysates were prepared with RIPA buffer to measure protein using the BCA Protein Assay Reagent (Thermo Fisher Scientific).