Extended Data Figure 1. FACS gating strategy for resolving basal and luminal subsets from mammary tumors.

Mammary tumors were mechanically and enzymatically dissociated into single cell suspensions. a, Negative selection against Lin⁺ cells using Stem Cell Technologies EasySep Mouse Epithelial Cell Enrichment Kit. Resulting Lin⁻ (CD45⁻/ CD31⁻/ TER119⁻/ BP-1⁻) cells were then immunostained with antibodies for CD49f (α6 integrin) and EpCAM and analyzed by FACS. b, Exclusion of cell debris and dead/ dying cells. Dead/dying cells collect as a band along the bottom of a FSC-A vs. SSC-A two-parameter plot, and these were gated out in P1. c, Cell doublets were discarded in P2. d, Basal and Luminal mammary epithelial cell populations were separated by immunophenotype. Basal epithelial cells are CD49f^high/ EpCAM^low (P3) and luminal epithelial cells are CD49f^Low/ EpCAM^high (P4). e, Gating tree showing gating strategy for FACS analysis as well as parent and total cell percentages within each of the gates for a representative MMTV-Wnt1 tumor.