Nu serum 1 2.5

HEK293 cells were purchased from the American Type Culture Collection (ATCC, VA, USA) and cultured at 37°C under a 5% CO₂ atmosphere in a humidified incubator. A phenol red-free version of Dulbecco’s Modified Eagle’s Medium (DMEM/High Modified) (Hyclone, GE Lifesciences, MA, USA) was used as the culture medium. One percent penicillin/streptomycin (Corning, NC USA) and 10% dextran-coated charcoal-treated fetal bovine serum (DCC-FBS; Atlanta Biologicals, MN, USA) were supplemented for all cultures. Fifty micrograms Zeocin/ml (Thermo Fisher Scientific, MA, USA) and 400 μg G418/ml (Calbiochem, MA USA) were supplemented during clonal selection of cell lines. Twenty-five micrograms Zeocin/ml and 100 μg G418/ml were supplemented for routine maintenance after selection. No antibiotics were supplemented during compound testing. Human adrenocortical carcinoma H295R cells were purchased from ATCC and cultured at 37°C and 5% CO₂ in a humidified incubator. DMEM-F12 medium (Gibco, MA, USA) was used as the culture medium. Nu-Serum I 2.5% (Corning) and 1× ITS+Premix (Corning) were supplemented for all cultures. Before use in steroidogenesis assays and or freezing, cells were maintained for five passages.