Breast Cancer Cell Lines Maintenance

The MDA-MB-231, SKBR3 and MCF12A cell lines were purchased from the American Tissue Culture Collection (ATCC) and the MCF7 cell line was acquired from the European Collection of Authenticated Cell Cultures (ECACC). MCF12A is a non-tumor breast cell line, while the others are tumor cell lines representative of different BC subtypes: MCF7—Luminal A; SKBR3—HER-2 subtype; MDA-MB-231—TNBC [88 (link),89 (link)]. MCF7, SKBR3 and MDA-MB-231 cells were cultured in high glucose DMEM deprived of phenol red and supplemented with 10% of FBS and 1% of the streptomycin-penicillin solution. MCF12A cells were cultured in DMEM/F12 supplemented with 20 ng/mL of EGFR, 100 ng/mL of cholera toxin, 0.01 mg/mL of human insulin and 500 ng/mL hydrocortisone, 10% FBS and 1% of streptomycin-penicillin solution. All the cell lines were maintained as monolayer cultures in T75 cm² culture flasks (Orange Scientific, Belgium) and incubated under standard cell culture conditions (37 °C, 5% CO₂). When reaching approximately 80% confluence, cells were subcultured using 0.25% trypsin/EDTA at 37 °C, counted in a hemocytometer and assessed for their viability using the standard trypan blue staining procedure. All experiments were conducted with cells at passages under 40.