2.5. Glucose-Stimulated Insulin Secretion (GSIS)

MIN6 cells were seeded on 24-well plates in amount of 2 × 10⁵ per well 48 h prior to the experiment. Confluent cells were initially pre-incubated for 60 min with a pH 7.4 calcium buffer pH 7.4 as previously [24], supplemented with 2 mM glucose. Subsequently, cells were incubated in the same buffers with tested compounds at either 25 or 50 µM working concentrations and/or GPCR antagonists (2 µM of DC, CID, and C8) for 30 min. The buffer samples were collected, and the same cells were incubated for another 30 min with fresh buffer supplemented with 20 mM glucose and respective test compounds. After collection of buffer samples from the high glucose conditions, cells were washed with cold PBS and lysed with 0.1 M HCl. Both the buffer samples and cell lysates were stored below −20 °C for further analysis. Buffer samples were used for insulin secretion measurements by competitive ELISA, as previously described [24]. Quantities of secreted insulin were normalized to protein contents in respective cell lysates measured according to Bradford Protein Assay.