Culturing Adrenocortical Carcinoma Cell Lines

NCI-H295R (RRID: CVCL_0458) and SW-13 (RRID: CVCL_0542) were purchased from ATCC. CU-ACC1 (RRID: CVCL_RQ00) and CU-ACC2 (RRID: CVCL_RQ01) were provided by Dr. K. Kiseljak-Vassiliades (5 (link)). NCI-H295R cells were grown in 1:1 DMEM:F12 (Thermo Fisher Scientific) supplemented with 2.5% Nu-Serum (Corning), 1% ITS media supplement (R&D Systems), and 1% Penicillin–Streptomycin (Thermo Fisher Scientific). SW13 was grown in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS (GeminiBio) and 1% Penicillin–Streptomycin (Gibco). CU-ACC1 and CU-ACC2 ACC cell lines were grown in F medium [3:1 (v/v) Ham's F-12 Nutrient Mixture–DMEM (Thermo Fisher Scientific) supplemented with 5% FBS, 0.4 µg/mL hydrocortisone (Sigma-Aldrich), 5 µg/mL insulin (Sigma-Aldrich), 8.4 ng/mL cholera toxin (Sigma-Aldrich), 10 ng/mL EGF (Invitrogen), 24 µg/mL adenine (Sigma-Aldrich)] (5 (link)). All cell lines were cultured at 37°C in a humidified incubator with 5% CO₂. Cell line identification tests were performed by short tandem repeat polymorphism analysis, using previously reported standard methods (5, 26 (link)). All cell lines were tested by MycoAlert Mycoplasma Detection Kit (Lonza) and confirmed to be Mycoplasma negative.