Mammosphere formation assay

The mammosphere assay was developed as an approach to propagate mammary epithelial stem cells [47]. This assay was performed according to an online protocol (http://www.bio-protocol.org/e325). Briefly, tumorsphere medium was made by adding 20ng/ml epidermal growth factor (EGF), 10ng/ml basic fibroblast growth factor (bFGF), 5ug/ml Insulin and 0.4% Bovine Serum Albumin in DMEM/F12 (50/50) medium, and B27 supplement (Thermo Fisher) was freshly added to tumorsphere medium. Cells were collected, washed and counted followed by resuspending in tumorsphere medium with B27 supplement at a final concentration of 10,000 cells/ml. Then 2 ml cells were added to each well of an ultra-low attachment 6-well plate (Corning). After 7-day incubation, pictures of each well were taken and colonies were quantified using CellSens Dimension software. Secondary or tertiary mammospheres were generated by digesting primary mammospheres or secondary mammospheres and were seeded at the same density as primary mammospheres. All experiments were performed three times and bars represent the means of three independent experiments ± s.d.