Quantification of Breast Stem Cells

For cells suspension culture, cells were plated in 100 mm non-adhesion culture dish (JET BIOFIL) over 5 days, regularly change the medium and verified by ALDH1 expression through flow cytometry detection as above. ZNF32 and GPER expression detection in suspension or normal cultured cells were examined by flow cytometry (BD FACSAria, Franklin Lakes, NJ, USA), according to the manufacturer’s recommended protocol. ZNF32 antibody was produced and purified as previously described^{27 (link)}. GPER antibody was purchased from Santa Cruz Biotechnology (Santa, sc-48525-R #B0216 USA). Rhodamine (TRITC) -conjugated AffiniPure Donkey Anti-Mouse (H + L) antibody was purchased from BBI Life Sciences (#D1100883–0100, China). FITC Conjugated Goat Anti-Rabbit Secondary Antibody was purchased from BOSTER (#BA1105, China). Individual fluorescent populations were determined using acquisition and analysis software (FlowJo 7.6).
A detailed description of the mammosphere assay protocol for the quantification of breast stem cell activity is described in a recent publication^{3 (link),28 (link)}. Briefly, cells were plated in suspension culture at 500 cells/cm². Mammosphere forming was calculated by dividing the number of mammospheres (colonies > 60μm in diameter) formed by the number of cells plated and expressed as a number.