2.8. Insulin Secretion Assay

Insulin secretion assay was performed as we previously described [7, 8]. Briefly, Min6 cells (p38–41) were incubated with cholesterol in the presence or absence of SFN or ES for 6 h, in serum-free and glucose-free DMEM supplemented with 2 mM L-glutamine and 25 mM HEPES, pH 7.4. Cells were then washed twice with PBS and placed immediately in serum-free DMEM with low glucose (5 mM) or high glucose (25 mM) for 1 h and medium was collected for the GSIS assay, according to Suzuki et al. [24]. Insulin levels in the medium were measured using a Rat Ultrasensitive Insulin ELISA Kit according to the manufacturer's instructions. Basal insulin secretion was measured in the absence of glucose in Min6 cells exposed to the different treatments. All values were normalized to protein content.