Cell Culture of Mammary Epithelial and Breast Cancer Cells

Mammary epithelial, non-transformed MCF10A cells (ATCC CRL-10317) were grown in DMEM:F-12 (Dulbecco's Modified Eagle Medium:Nutrient Mixture F-12) (1:1) medium (Gibco, 11330057) supplemented with 5% Horse Serum (HS), 2 mM l-glutamine and 1% penicillin/streptomycin (Sigma-Aldrich, G6784), 10 µg/ml insulin (Sigma-Aldrich, I9278) and 0.5 µg/ml hydrocortisone (Sigma-Aldrich, H0888). Human Epidermal Growth Factor (hEGF, Sigma-Aldrich, E9644) was added freshly before the use of full medium (20 nm/ml). MCF7 cells and MDA-MB-231 cells (ATTC HTB-26) were grown in DMEM medium (Gibco, 11330057) supplemented with 10% Fetal Bovine Serum (FBS, Euroclone, ECS0180L), 1% penicillin/streptomycin (Sigma-Aldrich, G6784) and 2 mM l-glutamine. Cells were grown on 10 cm² petri dishes for a month (about 10–12 cell passages) at 37 °C in 5% CO₂. For any experiments on fixed cells, cells were plated on glass coverslips coated with 0.5% (w/v) pork gelatin (Sigma-Aldrich, G2500) previously dissolved in phosphate buffer saline (PBS) and autoclaved.