Ter119 bv421

Single-cell suspension of isolated primary mouse mammary epithelial cells (MMECs) was resuspended in 0.2% BSA in Dulbecco's PBS, and the cells were incubated with the following primary antibodies for analysis: CD29-FITC (Miltenyi Biotech, 130-102-975); CD24-APC (M1/69, BioLegend, 101814); CD31-BV421 (MEC13.3, BD Biosciences, 562939); CD45-BV421 (30 F11, BD Biosciences, 563890); Ter119-BV421 (BD Biosciences, 563998); Sca1-BV711 (D7, BioLegend, 108131); and CD49b-PE (HMα2, BioLegend, 103506). All antibodies were diluted 1:500 at 4°C for 30 min. Cells were washed with PBS and resuspended in 0.2% BSA in Dulbecco's PBS with 1:500 Sytox Blue (Life Technologies, Thermo Fisher Scientific) to exclude dead cells. Sorting was performed using a FACSAria Fusion cell sorter (Becton Dickinson). FlowJo V10 was used for post-analysis of sorted cells.

Peripheral blood counts were acquired using the V-Sight hematology analyzer (A. Menarini Diagnostic). RBCs were lysed for 5 min at RT in RBC lysis buffer. Freshly isolated BM, spleen, thymus, lymph node, and white blood cells were stained with Fixable Viability Dye eFluor 780 (eBioscience), blocked for 10 minutes with TruStain FcX anti-mouse CD16/32 (BioLegends) and stained for 30 minutes in brilliant stain buffer (BD Bioscience) with antibodies against Ly5.1-PerCP-Cy5.5 (eBioscence #45-0453), Ly5.2-V500 (BD Bioscence 562129), CD19-BV786 (BD Bioscience #563333), CD3e-APC (BD Bioscence #553066), Mac1-BV650 (BD Bioscience #563402), Gr-1-APC (eBioscience #17-5931) and Ter119-BV421 (BD Bioscence 563998). Cells were analyzed by FACSFortessa (BD BioScience) and FlowJo software.