Insulin elisa assay kit

EndoC-betaH1 cells were preincubated with GDF15 at the concentrations of 0, 10, or 1000 ng/mL for 24 h. The cells were then either left untreated or treated with a mixture of cytokines (20 ng/mL IL-1β + 20 ng/mL IFN-γ) or 1.5 mM palmitate + 22 mM glucose in the presence or absence of GDF15 for the next 24 h. Krebs-Ringer bicarbonate HEPES buffer (KRBH) containing 0.5% BSA and 1.7 mM glucose was added after the treatment medium was removed, and the cells were preincubated for 30 min at 37 °C. Thereafter, the cells were incubated with 16.7 mM glucose in KRBH + 0.5% BSA buffer at 37 °C for another 60 min. Insulin was then analyzed in cell supernatants collected from 16.7 mM glucose exposed cells. In order to measure the insulin content, cells were harvested and homogenized by sonication in distilled water. Samples were mixed (1:2) with 95% acid ethanol and kept for 2 h at −20 °C prior to supernatant separation. Insulin levels were then evaluated using the insulin ELISA assay kit (Mercodia, Uppsala, Sweden), and the Bradford protein assay was used to determine the total protein content.