4.5. Glucose-Stimulated Insulin Secretion (GSIS)

MIN6 cells were seeded in the amount of 2 × 10⁵ per well 48 h on 24-well plates. For the first 60 min, confluent cells were pre-incubated with a calcium buffer (25 mM HEPES, 125 mM NaCl, 6 mM KCl 1.2 mM MgCl₂:6H₂O, 1.3 mM CaCl₂:2H₂O, pH 7.4) supplemented with 2 mM glucose. Subsequently, cells were incubated in the new portions of the same buffer supplemented with LPCs or their corresponding acids at either 5, 10, or 25 μM working concentrations and/or GPCR antagonists (2 μM of DC, CID, C8, and AH) for 30 min. MIN6 cells were also stimulated with glucagon-like peptide (GLP-1) in which case 50 nM, 100 nM, 500 nM, 1 μM, or 5 μM concentrations were tested. The buffer supernatants were saved and the same cells were incubated for another 30 min with 20 mM glucose buffer supplemented with test compounds. After the collection of buffer samples from the high-glucose conditions, cells were lysed with lysis buffer and subjected to competitive ELISA [55]. The protein content of respective cell lysates was determined using the Bradford Protein Assay, and the amounts of secreted insulin were normalized.