Splenic HSPCs reside near VCAM-1⁺ macrophages. (A) Gating for endothelial cells. CD45.2⁻ CD31^high ICAM-2^high cells were considered as endothelial cells. (B) VCAM-1 expression on splenic endothelial cell and leukocytes by FACS. (C) Cluster of GFP⁺ cells in the splenic red pulp 3 d after GFP⁺ HSPC transfer into wild-type mouse. Bar, 15 µm. (D) Cluster of GFP⁺ cells (green) near VCAM-1⁺ macrophages (red). Bar, 20 µm. (E) GFP⁺ cells (green) in close contact with VCAM-1⁺ (red) macrophages (F4/80⁺, blue) in the spleen. Bar, 5 µm. (F) 3D image of interaction between a GFP⁺ cell and a VCAM-1⁺ macrophage. (G) Fraction of GFP⁺ cells within indicated distance from VCAM-1⁺ macrophages (n = 5). (H) We induced parabiosis of GFP⁺ and GFP⁻ mice. After 6 wk of parabiosis, the spleens of GFP⁻ wild-type mice were analyzed for chimerism (GFP⁺ cells) using flow cytometry (H) and immunofluorescence microscopy (I; n = 3). Bar, 10 µm. Data were pooled from two independent experiments. Data are mean ± SEM. Significance was determined by one-way ANOVA and Mann-Whitney test. *, P < 0.05; ***, P < 0.01.