Only CD163 ⁺ macrophages monitor the vasculature. (A) Flow cytometry quantification of BSA-A647 (4 mg/kg, 1.5 h) and IgG-A647 (6 mg/kg, 2 h) uptake in indicated organs/cell subsets. Gated on CD11b⁺Ly6C^hi (monocytes), CX3CR1^hiCD64^low (microglia), and CX3CR1⁺CD64^hi (macrophages). The experiment was performed two (IgG, n = 5 total) or three times (BSA, n = 8 total). Tukey’s multiple comparisons test. ***P < 0.001. (B) Machine-learning (DRGQuant)-based analysis of uptake of BSA-A647 (20 mg/kg, 1 h), goat anti rabbit IgG-A488 (4 mg/kg, 4 h) in CD163⁻Iba1⁺ and CD163⁺Iba1⁺ macrophages without (parenchymal) or with (perivascular) contact with vasculature. Perivascular macrophages were additionally analyzed based on which vessel segment they contacted. Student’s unpaired t test. ***P < 0.001. Scale bar, 25 μm. (C) Experiment schematic of tracer uptake in macrophage depleted mice using the CSF1R antagonist PLX3397 (290 ppm in chow). Created with https://BioRender.com. (D) Uptake of coinjected BSA-A647 (4 mg/kg), 70 kD dextran-TMR (10 mg/kg), and goat anti rabbit IgG-A488 (3 mg/kg) in Iba1⁺ macrophages 2 h 45 min after i.v injection. Depletion experiment was performed three times, uptake once. Student’s unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant. Scale bar, 50 μm. (E) Experiment schematic of tracer uptake in CD163⁺ macrophage depleted mice using αCD163 or isotype control antibody (2.5 mg/kg, three injections separated by 48 h). Created with https://BioRender.com. (F) Quantification of macrophage subsets and uptake of i.v injected BSA-A488 (4 mg/kg) and goat anti human IgG-A647 (3 mg/kg) 2 h after i.v injection. n = 9, 10 mice. Two experiments pooled. Depletion experiment was performed three times, uptake twice. Student’s unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant. Scale bar, 50 μm. (G) Flow cytometry UMAP of CD45⁺CD11b⁺Ly6C⁻CD64⁺CX3CR1⁺ macrophages after αCD163 mediated depletion (2.5 mg/kg, three injections separated by 48 h). n = 4 (isotype), 3 (αCD163).