Extended Data Fig. 1: Validation of stains to detect myeloid cells.

a, FACS plots showing the gating strategy used to identify MDPs and MOPs (as described in ref. ¹⁹). BM, bone marrow. b, Gating strategy used to identify GMPs, GPs and MOPs (as described in ref. ¹⁶). The lineage panel contains antibodies against Ly6G, CD11b, Ter119, B220 and CD3. c, Images showing that Ly6C labels arterioles, detected as either CD31⁺ CD144⁺ Sca1^bright in wild-type mice or CD31⁺ CD144⁺ Nestin^bright structures in Nestin–GFP mice; the histograms show quantifications demonstrating that all Sca1⁺ arterioles or Nestin–GFP^bright arterioles are also Ly6C⁺. Scale bars, 50 μm. d, e, FACS plots (d) showing that only the CD11b⁺ gate contains CD117⁺ CD115⁺ or CD117⁺ Ly6C⁺ cells, and histogram (e) showing that GPs and MOPs are the only Ly6C⁺ cells in the Lin⁻ CD117⁺ gate. Together these data indicate that CD11b alone can be used to replace the lineage panel to exclude contamination of mature cells when detecting MDPs, MOPs and GPs. f, Cell numbers per femur detected by FACS of the indicated progenitors using previously described strategies^16,19 (white) or that described in Fig. 1g (purple). g, Colony-forming activity (green, MDPs; orange, MOPs; red, GPs) of the indicated progenitors using previously described strategies^16,19 (diamonds) or the one described in Fig. 1g (circles). For f, g, n = a total of three mice in two experiments. h, Frequency of total BM cells for each of the indicated populations in sternum when detected by FACS (white) or imaging (orange). n = 3. i, Representative image showing that CD11b⁻ CD117⁺ CD115⁺ Ly6C⁻ MDPs and CD11b⁻ CD117⁺ CD115⁺ Ly6C⁺ MOPs are GFP⁺ in Cx3cr1–gfp mice. Scale bar, 10 μm. j, qPCR results showing Gfi1 and Irf8 expression (relative to Gapdh expression) in FACS-purified GPs or MOPs; n = a total of six mice in three experiments. k, FACS analyses in Gfi1–tdTomato mice showing differential tdTomato expression in GPs and MOPs in Gfi1–tdTomato mice. l, Quantification of promyelocytes (PMs), myelocytes (MCs), metamyelocytes (MMs), banded cells (BCs) and polymorphonucleated neutrophils (PMNs) in cytospin preparations of FACS-purified PNs, INs and MNs. n = a total of two mice. Scale bar, 10 μm. m, The stains require discrimination of CD16/32, CD117 and Ly6G bright and dim cells. The panels show the gating strategy, experimental design, and quantification of frequencies of decanted CD16/32 and CD117 bright and dim cells or INs, PNs and MNs when compared with frequencies obtained by FACS before cytospinning. Each dot represents one image field from two experiments. n, o, Dendritic cells can be imaged as reticulated CX3CR1–GFP⁺ or CX3CR1–GFP⁺ MHC II⁺ cells in Cx3cr1–gfp reporter mice^23,24. The images and histograms show that all reticulated GFP⁺ cells were also MHC II⁺ and CD11c⁺, indicating that MHC II expression and cell shape are sufficient to unambiguously identify dendritic cells and distinguish them from macrophages that are CXCR1–GFP⁻ CD11c⁻ cells²⁴; n = a total of three mice. Scale bar, 10 μm. p, Image and histogram showing that CX3CR1–GFP⁺ MHCII⁺ dendritic cells are conventional dendritic cells, as they are CD11b⁺ but do not express B220 or CD8. Scale bar, 10 μm. n = a total of three mice. q, FACS gating strategy for isolation and imaging of the indicated cells. Dendritic cells are detected as MHCII⁺ reticulated cells in imaging analyses. In all bar graphs, one dot corresponds to one mouse. Statistical differences were calculated using two-tailed Student’s t-tests; P values are shown.

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