Immortalized Breast Cell Culture

Human mammary immortalized cells (MCF10A), and the TNBC cell line MDA-MB-231 were purchased from American Type Culture Collection. MCF10A cells were grown in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F12 medium with 20 ng mL⁻¹ human epidermal growth factor, 100 ng mL⁻¹ cholera toxin, 0.01 mg mL⁻¹ bovine insulin, 500 ng mL⁻¹ hydrocortisone, and 5% horse serum. MDA-MB-231 cells were grown in DMEM containing L-glutamine and sodium pyruvate, supplemented with 10% fetal bovine serum and 1% antibiotic and antimycotic solution in a humidified atmosphere of 5% CO2 at 37°C in an incubator. Human BCSC cells: ALDH⁺ and CD44⁺/CD24⁻, and BC cells: ALDH- were purchased from Celprogen (San Pedro, CA, United States) and maintained in human BCSC expansion and undifferentiation media. DAPT (γ-secretase), cycloheximide (CHX), chloroquine (CQ), and MG132 were purchased from Sigma (St. Louis, MO).