Insulin transferrin selenium ethanolamine its

Mouse Y1 cells and human NCI H295R adrenocortical cells were seeded into 12-well plates and incubated overnight using Dulbecco’s modified Eagle’s medium high glucose (4.5 g/liter) (Gibco) with 7.5% horse serum (Gibco), 2.5% fetal bovine serum (FBS) (Gibco), and 1% penicillin-streptomycin (Gibco) and RPMI 16/40 + GlutaMax (Gibco) with 10% FBS (Gibco), 1% Insulin-Transferrin-Selenium-Ethanolamine (ITS) (Thermo Fisher Scientific), and 1% penicillin-streptomycin (Gibco), respectively. In this experiment, 100,000 Y1 and NCI H295R adrenocortical cells per well were used. Cells were then stimulated for 24 hours with 10 nM forskolin and subsequently treated with different concentrations of tariquidar (0, 10, 50, 125, 250, 500, and 1000 nM) and incubated for 24 hours. Last, supernatants and cell pellets were collected and harvested for further analyses and measurement of CORT (ng/ml) and cortisol (μg/liter) levels. Media CORT levels in Y1 cells were quantified by RIA using a CORT double antibody 125I RIA kit, as previously described in the animal experiments. Media cortisol levels in NCI H295R adrenocortical cells were determined using an enzyme-linked immunosorbent assay (ELISA) kit (RE52061, TECAN, IBL Hamburg, Germany). The standard range was 20 to 800 ng/ml.