Culturing and Analyzing Breast Cancer Cells

Cancer cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). We followed a previously described method [18 (link)] and the breast cancer cells were incubated in Dulbecco’s modified essential medium (DMEM; Hyclone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 1% penicillin/streptomycin (Hyclone, Logan, UT, USA). Breast cancer cells were incubated at 37 °C in 5% CO₂ incubator. Cancer cells were plated with 2 × 10⁶ cells in 10 cm culture dishes. For mammospheres culture, cancer cells were cultured with 3.5 × 10⁴ and 0.5 × 10⁴ cells/well with MammoCult^TM medium (StemCell Technologies, Vancouver, BC, Canada). The cells were incubated in a 5% CO₂ incubator. The complete MammoCult™ medium was supplemented with 4 μg/mL heparin, 0.48 μg/mL hydrocortisone, 100 U/mL penicillin, and 100 μg/mL streptomycin. At 7 days of culture, a 6-well plate was scanned and counted using the software program NICE [19 (link)]. For mammosphere formation assay, MFE (%) was determined as previously described [20 (link)].