Cd29 clone hmβ1 1

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)].

Bone marrow-derived DCs were cultured in CM ± freshly egressed T. gondii tachyzoites (ME49/PTG-GFP, MOI 1) or LPS (100 ng/ml, serotype 011:B4, Sigma-Aldrich) for 4 or 24 h. Cells were stained on ice in FACS buffer (1% FBS and 1 mM EDTA in PBS) with Live/Dead Violet (L34955, Life technologies), anti-CD11c (clone N418, 25-0144-82, eBioscience) and anti-CD18 (clone M18/2, 101407, BioLegend), -CD29 (clone HMβ1-1, 102213, BioLegend) -CD54 (clone YN1/1.7.4, 116119, BioLegend) or isotype control antibodies (clone R35-95, 553930, BD Pharmingen; clone HTK888, 400924, BioLegend; clone RTK4530, 400611, BioLegend), fixed with 2% PFA and analysed on a BD LSRFortessa flow cytometer (BD Biosciences) with FlowJo software (v. 10, FlowJo LLC). Prior to staining, cells were blocked in FACS buffer supplemented with anti-CD16/CD32 antibody (Fc Block, BD Pharmingen).