Flow Cytometry Isolation of Mammary Cancer Cells

MDA-MB-231, MCF7, and SKBR3 human cancer cell lines were trypsinized to single cells, and MMTV-NDL murine mammary tumors (1.0–1.5 cm in diameter) were harvested and dissociated to single cells as previously described [29 (link),30 (link)] with minor modifications. Cells were suspended at 1 × 10⁷ per mL in staining buffer (PBS with 2% FBS) and incubated for 30 min on ice with antibodies. Cells were washed three times, re-suspended in staining buffer with 1 μg/mL Propidium Iodide (PI), analyzed, and sorted with a FACS Aria II cell sorter (Becton Dickinson, Franklin Lakes, NJ, USA). Sorting schemes were based on previously published studies for human cell lines [1 (link),8 (link)] and primary mouse tissues [33 (link),34 (link)]. Results were analyzed using FlowJo software. Antibodies used were CD24-PE-Cy7 (1:100 dilution, #561646) and CD44-APC (1:100 dilution, #559942; BD Pharmingen, San Diego, CA, USA) for human cells, and CD24-PE (1:200 dilution, #553262; BD Pharmingen), CD49f-APC (1:100 dilution, #313615; Biolegend, San Diego, CA, USA), CD31-PE-Cy7 (1:100, #102417; Biolegend), and CD45-PE-Cy7 (1:100, #103113; Biolegend) for mouse cells.