Figure 8.

Gating strategy for flow cytometry analysis and sorting of mouse mammary epithelium

(A) Cells were gated for forward (FCS-A) and side (SSC-A) scatter to remove debris and population p1 was selected.

(B and C) (B) P1 from (A) was plotted on FSC-H/A and single cells (p2) were selected and then gated (C) to remove dead cells (Zombie Yellow positive) and Lineage⁺ (Cd140a⁺; CD31⁺; Ter-119⁺; and CD45⁺) cells (Live lineage-).

(D) Live cells from (C) were plotted on EpCam and CD49f and the luminal population (EpCam^high CD49f^medium) was selected.

(E and F) (E) Luminal cells from FMO controls (tube 10: FMO-Sca1; tube 11: FMO-CD49b) were plotted on Sca-1/CD49b to determine the 4-way gates. Gates from (A)–(D) were reapplied to tube 12, and (F) luminal cells from (D) were separated into CTV+ and CTV- populations.

(G) 4-way gates from (E) were reapplied to CTV+ and CTV- cells from (F) to perform 4-way split.