Truestain fcx antibody

Mouse PBMCs were incubated with True Stain FcX antibody (clone 93, BioLegend) in PBS with 2% FBS before staining with antibodies directly conjugated to fluorophores. Cells were divided into two tubes to stain for myeloid markers (CD11b, CD11c, Ly6C, Ly6G, MHC I, MHC II) or lymphoid markers (CD3, CD4, CD8, CD19, c-kit). Antibodies are summarized in Supplementary Table 2. DAPI was used to exclude dead cells from analysis. Cells were washed and strained through a 40 μm filter (BD Falcon, 352340) after staining and analyzed on a BD LSRII flow cytometer. AbC Total Antibody Compensation Bead Kit (Thermo Fisher, A10513) was used for single color compensation. Flow cytometry data were then analyzed using FlowJo software (version 10.8.0, Becton Dickinson) to quantify the frequency of cells positive for each marker; the gating strategy is outlined in Supplementary Fig. 6. Lymphoprep centrifugation is selective for T, B, NK, and monocyte populations and excludes granulocytes, consistent with the observed low abundance of those myeloid markers (e.g., Ly6G+) in the cells analyzed. Percentages (SAFE, FLOW) of positive cells for each marker are c-Kit (0.5%, 0.9%), CD8 (13.7%, 13.1%), CD11c (2.1%, 3.2%), CD19 (66.9%, 55.8%), CD45 (98.6%, 99.1%), CD11b (6.5%, 11.9%), CD4 (16.2%, 9.9%), CD3 (30.8%, 25%), MHCII (72.3%, 66.1%), MHCI (22.1%, 23.7%), Ly6G (0.0%, 0.2%), Ly6C (11.6%, 7.7%).