Hematopoietic stem and progenitor cell identification and quantification by flow cytometry

Mouse bones (femur and tibia) were harvested, crushed in PBS + 2%FBS and filtered through a 0.7 µm mesh. Bone marrow (BM) cells ~10⁶ cells were stained with a lineage cocktail against biotinylated CD3, CD4, CD8a, Gr-1, B220, Ter-119, and CD11b and identified using streptavidin Pacific Blue™. Within the lineage negative fraction, stem/progenitor populations were assessed using antibodies against c-Kit and Sca-1 (Lin⁻c-Kit⁺Sca-1⁺ (LSK) and Lin⁻c-Kit⁺Sca-1⁻ (LK) cells). To assess the frequency of progenitor cells within the lineage negative fraction, c-Kit and Sca-1 staining, in addition to CD127, CD34 and CD16/32 was used to identify CMP (CD34⁺ CD16/32⁻), MEP (CD34⁻ CD16/32⁻), and GMP (CD34⁺ CD16/32⁺) cells⁶⁰. All antibodies were purchased from BD Biosciences, Biolegend, eBioscience or Life Technologies. The percentage of various cell populations within the bone marrow (BM) was examined and the percentage of the parent gate and percentage of the total BM was reported (Supplementary Fig. ^S5). Cellularity was assessed (Hemovet®) and white blood cell count was multiplied by the white percentage positive cells within total BM to give absolute numbers per harvest. Flow cytometry analysis of myeloid and lymphoid mature cells was performed on BM, spleen, peripheral blood (PB) and thymi, which were prepared in PBS + 2%FBS and ~10⁵ cells were stained against markers of myeloid (Gr1, CD11b), lymphoid (CD19), erythroid (Ter119) or T cells (CD4 and CD8a). T cells were identified as naive or memory based on the cell surface staining of CD44 and CD45RB. PB was collected into EDTA-coated blood tubes (Sarstedt AG & Co) and stained with the appropriate antibody cocktail. After incubation, blood was lysed using EasyLyse (Dako UK Ltd) and subsequently analysed.