Cd49b pe hmα2

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.

Mouse mammary cells were preblocked with 10% normal rat serum and then incubated with the following primary antibodies: CD31-biotin (clone 390, eBioscience), CD45-biotin (clone 30-F11, eBioscience), Ter119-biotin (clone Ter119, eBioscience), BP-1-biotin (clone 6C3, eBioscience), EpCAM-APC (clone G8.8, BioLegend), CD49f-AF488 (clone GoH3, BioLegend), CD49b-PE (HMα2, BioLegend), and Sca1-PE/Cy7 (clone D7, BioLegend). In some experiments, cells were stained with CD24-PE (clone M1/69, BioLegend) and CD29 (clone HMβ1-1, BioLegend). CD45, Ter119, CD31, and BP-1 were used to deplete contaminating hematopoietic, endothelial, and a proportion of stromal cells, respectively (collectively termed Lin + cells). Biotin-conjugated antibodies were detected with Streptavidin-APC-Cy7 (BioLegend). Cells were then filtered through a 30-μm cell strainer and incubated with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) and were analyzed by using an LSRII (Becton Dickinson) and were sorted on a FACSAria I (Becton Dickinson). The flow-cytometry gating strategy was as previously described [11 (link)].

Splenocytes were stained with fixable viability dye eFluor780 (1:1,000) before staining for surface antigens CD4-A700 (GK1.5, 1:100), CD69-PE-Cy7 (H1.2F3, 1:300), CD62L-PerCP-Cy5.5 (MEL-14, 1:200), LAG-3-PE (C9B7W, 1:200), TIM-3-PE (8B.2C12, 1:200) (all eBioscience) and PD-1-PE-Cy7 (29F.1A12, 1:300), TIGIT-APC (1G9, 1:200), CD49b-PE (HMα2, 1:100) (all Biolegend). For staining of transcription factors and Ki67, cells were then fixed with fixation/permeabilization buffer and antibodies were diluted in permeabilization buffer (both eBioscience); Ki67-PE (SolA15, 1:200), c-Maf-eFluor660 (SYMOF1, 1:200), NFIL3-PE (S2M-E19, 1:200), FoxP3-eFluor450 (FJK-16S, 1:100). Data were collected using an LSR II flow cytometer (BD Biosciences) and analysed using FlowJo software (Treestar).