Cd49f and cd24

For isolation of MaSC and BCSCs, we used CD24^lo and CD49f^hi as markers for normal MaSC and CD44/CD24 double-positive for human BCSCs and CD49f/CD24 double-positive for mouse BCSCs as described in our previous manuscripts (15 (link), 21 (link)) and studies from other laboratories (22 (link), 23 (link)). We used the following antibodies for this study: CD49f and CD24 (STEMCELL Technology and eBioscience), CD45, CD31, CD49f, CD44 (BD bioscience), and Ter119 and CD61 (eBioscience). Blocking was done for 10 min with rat serum. Cells were stained for 30 min on ice and washed with staining media. Finally, cells were resuspended in staining media containing 7-aminoactinomycin D (1 μg/ml) or 4’−6-diamidino-2-phenylindole (DAPI, 1 μg/ml) to stain dead cells. Cells were sorted through Mo flow cell sorter and flow cytometry data was analyzed using LSR II and Flow-jo as described before in our manuscripts (15 (link), 21 (link)).

For isolation of stem/progenitor cells, the following antibodies were used: CD49f and CD24 (Stem cell technology, eBioscience), CD45 and CD31 (BD bioscience), CD44 and ISL1 (BD bioscience), and Ter119 and CD61 (eBioscience). Blocking was done for 10 min with rat serum. Cells were stained for 30 min on ice and washed with staining media. Finally, cells were resuspended in staining media containing 7-aminoactinomycin D (1 µg/ml) or 4'-6-diamidino-2-phenylindole (DAPI, 1 µg/ml) to stain dead cells. Cells were analyzed using a LSR II, Flow-jo, and sorted Mo flow cell sorter. For flow cytometric analysis of ISL1 protein expression, CAL51-pCDH and CAL51-ISL1 stable cell lines and HBL-100, MCF10A, MDAMBA231, and MDAMB436 cells were fixed with BD Cytofix fixation buffer and permeablized with BD Phosflow perm buffer. Isl1-PE (BD Bioscience) conjugated antibody was used.