Proliferation and Insulin Secretion of EndoC-βH1 Cells

The EndoC-βH1 human beta cell line was cultured consistent with conditions described by Tsonkova et al [11 (link)]. The EndoC-βH1 was cultured in DMEM low glucose (1g/L), 2% Albumin from bovine serum fraction V, 50 μM 2-mercaptoethanol, 10 mM nicotinamide, 5.5 μg/ml transferrin, 6.7 ng/ml sodium selenite and Penicillin (100 units/ml) / Streptomycin (100 μg/ml) and maintained in sub confluent densities. EndoC-βH1 cells were seeded at 2 ×10⁵ cells per well in a cell culture treaded 96-well plate and allowed to adhere for 24 hours prior to starting of the experiments. The next day, seeding media was exchanged for fresh media containing 10% human serum from pregnant or non-pregnant donors together with EdU (Click-iT^™ EdU Proliferation Assay for Microplates, ThermoFisher Scientific) and cultured for 1 week. At takedown, EdU was detected following the kit manufacturer’s protocol and fluorescence was measured with a microplate reader (Molecular Dives Spectramax M5) at 568/585 nm. Secreted insulin in cell culture supernatant was measured by human Insulin Chemiluminescence ELISA kit (ALPCO).