Fig 1: Validated flow cytometry gating strategy for murine myeloid cells, related to Figure 2(A) CITE-seq data from the murine myeloid cells in Figure 2A were exported as an FCS file and an in silico gating strategy identified in FlowJo software.(B) Application of the in silico gating strategy with a 21-color flow cytometry panel. Myeloid cells were pre-gated as live CD45+ lineage cells (Ly6G-CD19-NK1.1-B220-CD3-). Data are representative of 3 experiments with 3–6 mice per experiment.(C) cDC1s, cDC2s, migratory cDCs (Mig. cDCs), peritoneal macs (Peri. Macs), KCs, and non-KC macs (non-KCs) were FACS-purified using gating strategy in (B), mRNA was isolated and qPCR performed to examine expression of indicated genes defining each population to validate their identity. Data are representative of 2 experiments with n = 3–6.(D) Putative peritoneal macs were FACS-purified using gating strategy in (B) and expression of Gata6 was examined by qPCR compared with other hepatic myeloid populations. Data are from a single experiment with n = 6.(E) Peritoneal macs as a % of total macs recovered from the liver using different digestion techniques (in vivo, ex vivo, or capsule) or in supernatants in which livers were washed following removal from the mouse but prior to digestion (wash). Data are from a single experiment with n = 4. *p < 0.05, **p < 0.01 one-way ANOVA with Bonferroni post-test compared with wash data.(F) Expression of CD14 and CD207 within the non-KC mac population from (B) (left) and % of CD207+ and CD207- populations among total macs in livers digested using the ex vivo or in vivo protocols or in dissected and digested liver capsule (right). Data are representative of two experiments with n = 4–5 mice per experiment. ****p < 0.0001 mixed effects analysis with Tukey’s multiple comparison test.(G) Expression of VSIG4, F4/80, GLUL, and DAPI by confocal microscopy. Insets represent zones featured in Figures 2E, 2G, and S3I.(H) Molecular Cartography of indicated genes and cell types. Insets represent zones featured in Figures 2F, 2H, and S3J.(I) Expression of VSIG4, F4/80, GLUL, and DAPI by confocal microscopy at the central vein. Scale bars, 50 µm.(J) Molecular Cartography of indicated genes and cell types at central vein.(K) Expression of F4/80, EPCAM, CCR2, GPNMB, and DAPI by confocal microscopy at a portal vein (top) or F4/80 or GPNMB alone (bottom). Scale bars, 25 µm.(L) Quantification of % of Gpnmb & Trem2 counts over Adgre1 counts in indicated regions of tissue as assessed using Molecular Cartography data. Each dot represents an individual region. *p < 0.05, ****p > 0.0001 one-way ANOVA with Bonferroni post-test.(M) Expression of DESMIN and F4/80 at the liver capsule and underlying parenchyma (left) or EPCAM, DESMIN and F4/80 at the bile duct by confocal microscopy. PV, portal vein; CV, central vein; HA, hepatic artery; BD, bile duct. Arrows indicate specific cell types, where color corresponds to cell type/markers. All images are representative of 2–6 mice.