Quantifying Insulin Secretion in β-Cells

Insulin secretion was quantified as the cumulative insulin release across each treatment period. The INS-1E β-cells and rat primary β-cells were prepared in 12-well plates (Corning) and exposed to KREBS solution containing 2.8 mM glucose for 30 minutes as the preincubation condition. Subsequently, the cells were incubated: (i) for 30 minutes under 2.8 mM glucose, (ii) for 5 minutes under 16.7 mM glucose, and (iii) for 30 minutes under 16.7 mM glucose conditions, respectively. The supernatant was aspirated at the respective end time point for each condition. Each sampling well was treated as an independent sample. After the supernatant was removed, cells were washed with 1xPBS once and detached by trypsin. Following centrifuge at 1000 rpm for 3 minutes, cells were resuspended in 1 mL of complete culture medium and then counted. Insulin levels in the supernatant were measured using an insulin ELISA kit (Mercodia). The protein content was subsequently evaluated using Flexstation 3.0 (Molecular Devices) with SoftMax Pro (v5.4.5.000, Molecular Devices). Finally, insulin secretion data were analyzed using GraphPad Prism (v9.4.1, GraphPad Software).