Fig 2: DC sortingMyeloid enriched splenocytes were incubated with live/dead stain, Fc-block and antibodies against CD11c and MHCII. DCs were sorted as alive single CD11c+MHCII+ cells to a purity >90%.

Fig 3: A population of macrophages reside around the bile duct in the healthy murine liver(A) UMAP of murine myeloid cells (71,261 cells/nuclei) isolated from Figure 1B and re-clustered with TotalVI.(B and C) Top DEGs (B) and DEPs (C) between cell types.(D) Expression of Gpnmb and Cd207.(E) Expression of VSIG4 and F4/80 (left) or MHCII, CD11c, and DAPI (right) by confocal microscopy. Capsule macs identified by white arrows. Scale bars, 50 µm.(F) Molecular Cartography of indicated genes at liver capsule.(G) Expression of VSIG4, F4/80, GLUL, and DAPI (left) or F4/80 or CCR2 (right, inset) by confocal microscopy. Scale bars, 100 µm.(H) Molecular Cartography of indicated genes at portal triad. PV, portal vein; CV, central vein; HA, hepatic artery; BD, bile duct. Arrows indicate specific cell types, where color corresponds to markers. Images are representative of 2–4 mice.(I and J) Top GO terms for KCs (I) and bile-duct LAMs (J).(K) Representative image showing expression of VSIG4 (red) CD19 (yellow) and CD3E (magenta) by MICS analysis (left) and % of B or T cells found with/without a KC per field of view (right). Data are pooled from multiple fields of view in 2 mice. ***p < 0.001 Student’s t test.(L) Mice (29-week-old) were treated with 3.5 mg/kg LPS or PBS and 2 h later, livers were harvested without the capsule. KCs and LAMs were FACS-purified and expression of Il1b, Tnf, IL10, and Il18 was examined by qPCR, compared with b-actin. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Bonferroni post-test. See also Figure S3 and Table S2.

Fig 4: Total-Seq-A antibody validationSplenocytes were incubated with flow cytometry live-dead stain, Fc-block and antibodies against MHCII and CD11c, and each Total-Seq-A antibody which was conjugated to a dT-oligonucleotide tagged fluorescence dye. Flow cytometry and Total-Seq-A antibodies against CD11c were specific to different epitopes.

Fig 5: Expected outcome of a bioinformatic unbiased analysis of the 16-color murine myeloid flow cytometry panel(A) Single alive CD45+ CD3- CD19- cells were subjected to bioinformatic FlowSOM analysis based on expression of Ly6G, Ly6C, PDCA1, CD11b, CD11c, F4/80, MHCII, CD172a, CD24, XCR1, and CD4 with a metacluster number of 40. User-determined thresholds for positive signal were applied to all features. The analysis identified 40 distinct clusters of cells.(B) Clusters were annotated based on key myeloid lineage features.