We used a highly differentiated rat insulinoma cell line (S5 cells) that was subcloned from the INS-1E cells. The cells were cultured in RPMI-1640 medium supplemented with fetal bovine serum (2.5% v/v), penicillin (50 IU/ml), 2-mercaptoethanol (500 µM), HEPES (10 mM), streptomycin (50 µg/ml) and sodium pyruvate (1 mM). The cells were cultured and maintained in humidified incubator in 5% CO₂ at 37⁰C.