Fig 2: Identification and characterization of macrophages in mouse spleen.a. Immunofluorescence showing GFP fluorescence (green, top left), MARCO staining for MZM (cyan, top right), CD169 staining for MMM (magenta, bottom left), and merge of all three channels (bottom right) from Cx3cr1gfp/wt mouse spleen. Scale bars 200 μm.b. Gating strategy for flow cytometry cell sorting used to isolate cells for cytospin analysis, starting with live singlets.c. Representative cytospin images. Scale bars 10 μm.d. UMAP plot showing integrated analysis of 12,114 myeloid cells from 3–4-month-old Cx3cr1gfp/wt mice, 2 male and 2 female.e. Expression of Cd163, Marco, Siglec1, and Gpnmb in the myeloid cells on the UMAP in d.f. Heatmap showing expression of selected genes within splenic macrophage clusters. Each row represents a single mouse.g. Immunofluorescent microscopy showing endogenous ZsGreen signal (green, top left), MARCO staining for MZM (cyan, top right), Tim-4 staining of MMM, MZM, and TBM (magenta; bottom left), and merge (bottom right) from Cx3cr1ERCre x ZsGreen mouse spleen. Scale bars 150 μm.

Fig 3: MMM and MZM are tissue-resident macrophages, MMM serve as a precursor population to MZM, and TBM have a dual ontogeny.a. Schematic of the lineage-tracing experiment. Tam: tamoxifen.b. Scatterplots showing percentage of ZsGreen-positive Tim-4+ macrophages in Cx3cr1ERCre x ZsGreen mice after tamoxifen treatment. Number of mice (from left to right): Female: 5, 5, 15, 6. Male: 13, 11, 17, 20. Results were compiled from 4 independent experiments. Significance was determined using one-way ANOVA, using a non-parametric Kruskal-Wallis test with Dunn’s multiple comparison test. The exact adjusted p-value is shown.c. Representative immunofluorescence microphotographs illustrating endogenous ZsGreen fluorescence (green), MARCO staining for MZM (cyan), CD169 staining for MMM (magenta), and merge of all three channels in the spleen of lineage-tracing Cx3cr1ERCre x ZsGreen mouse after tamoxifen treatment. Scale bars 200 μm.d. Schematic of the spleen transplant experimental design.e. Bar plot showing percentage of donor-derived total immune cells (CD45+ cells), RPM, and Tim-4+ macrophages (Tim-4 macs) 3 months post-transplant. Data from 2 experiments, a total of 5 mice, male mice were used for both donors and recipients. Kruskal-Wallis test followed by Dunn’s post-hoc testing was used to identify statistically significant differences between groups: adjusted p-value is 0.019 for CD45+ cells vs. RPM comparison, 0.029 for CD45+ cells vs. Tim-4+ macrophages, and >0.99 for RPM vs. Tim-4+ macrophages. ns: not significant.f. Schematic of the MMM depletion and repopulation experiment. Tam: tamoxifen. GM3-CL: GM3-modified clodronate-loaded liposomes.g. Representative immunofluorescence microphotographs illustrating endogenous ZsGreen fluorescence (green), MARCO staining for MZM (cyan), CD169 staining for MMM (magenta), and merge of all three channels in the spleen of lineage-tracing Cx3cr1ERCre x ZsGreen mouse after treatment with GM3-CL. Scale bars 200 μm.h. Box plot illustrating the change in percentage of ZsGreen/CD169 double-positive area after GM3-CL treatment. One-way ANOVA followed by Dunnett’s multiple comparison test was used to identify statistically significant differences between all groups.i. Box plot illustrating the change in percentage of ZsGreen/MARCO double-positive area after GM3-CL treatment. One-way ANOVA followed by Dunnett’s multiple comparison test was used to identify statistically significant differences between all groups.