Fitc and pe

Normal human mammary epithelial cells (MECs) were isolated from reduction mammoplasty specimens, and the resulting epithelial organoids were grown in matrix-embedded culture, as described previously [19 (link), 56 (link)]. After 9 days of culture, the acini were harvested, dissociated [20 (link)] and stained with specific cell markers. For MUC1/CD10 cell sorting experiments, single primary MECs from 3D cultures were stained with anti-MUC1 conjugated with fluorescein isothiocyanate (FITC; BD Biosciences) and anti-CD10 conjugated with phycoerythrin (PE; BD Biosciences). Single primary MECs were also sorted into subfractions on the basis of EpCAM, CD49f, CD10, MUC1, Thy1 and CD133, as described in [20 (link)]. For all flow cytometry experiments, forward scatter and side scatter plots were used to account for debris and aggregated cells. Single stained samples were used as compensation controls, and control samples consisted of unstained cells and/or IgG antibodies directly conjugated with FITC and PE (BD Biosciences). Cells were sorted on a FACSAria III cell sorter (BD Biosciences) in the Flow Cytometry Centre at the Westmead Millennium Institute. Analysis was performed using FACS Diva software (v6.1.2, BD Biosciences).

After a 15-min staining period at room temperature in the dark using FITC and PE (BD Biosciences), the cells were analyzed by flow cytometry within 1 h (BD Biosciences). Cell apoptosis analysis was performed using FlowJo software (BD Biosciences).