Multiparametric Flow Cytometric Analysis of Hematopoietic Subsets

Single cell suspensions were stained with three separate antibody cocktails for analyses of BM, spleen, and thymus. BM subsets were identified using the following conjugated mAbs: Ter119, CD25 (Invitrogen, Thermo Fisher Scientific, Waltham, MA), c-kit, Sca1, CD19 and CD44 (BD Biosciences, San Jose, CA). Lineage-differentiated BM cells were eliminated using a dump consisting of mAbs against CD11b, Gr1 and CD3. HSCs, MPPs, and HPCs were identified within the lineage^- Sca1⁺c-kit⁺ (LSK) lineage as a function of CD150 and CD48 profiles while CMP, GMP, and MEP were evaluated on lineage^-c-kit⁺ (LK) progenitors as a function of CD34 and CD16/32 expression (36 (link), 38 (link)). For thymus, cells were stained with the following directly conjugated mAbs: CD3, c-kit, CD4, CD8, CD44, TCRγδ (BD Biosciences) and CD25 (Invitrogen). Non-T lineage cells were excluded using a dump consisting of mAbs against CD19, Gr1, CD11b. Splenic subsets were distinguished using the following directly conjugated mAbs: Ter119 (Invitrogen), CD44, CD3, CD4, CD8, CD62L, TCRγδ, CD19 and CD11b (BD Biosciences). Stained cells were analyzed by flow cytometry using the LSR II-Fortessa (BD Biosciences). All data analyses were performed using Diva (BD Biosciences), and FlowJo Mac v.10.6.2 software (Tree Star).