Human insulin chemiluminescence elisa kit

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).

Study data were collected and managed using REDCap electronic data capture tools hosted at the University of Florida. After written, informed consent for participation was obtained, maternal peripheral blood samples were collected just prior to delivery. Centrifugation was performed to obtain serum and plasma samples (2000 × g at 4°C for 15 min). Serum and plasma samples were stored at −80°C until analyzed. Baseline blood glucose and insulin concentrations were measured on all maternal serum samples using an Accu-Check Aviva handheld glucose meter and human insulin Chemiluminescence ELISA kit (ALPCO).