PMos uptake sickle RBCs in vivo. (A) Schematic representation of experimental design. WT mice or CD11a^−/− mice were transfused with DiI-labeled RBCs from sickle Townes or control HbAA mice. At 20 hours after transfusion, mice were injected IV with freshly prepared hemin and analyzed 4 hours later. (B) Representative dot plots comparing DiI⁺ PMos (CD45⁺CD11b⁺Ly-6G^-CD115⁺Ly-6C⁻) and CMo (CD45⁺CD11b⁺Ly-6G^-CD115⁺Ly-6C⁺) from WT mice. For the gating strategy used, see supplemental Figure 4B. (C) Frequencies of DiI⁺ circulating leukocyte subsets in WT mice (n = 8) and CD11a^−/− mice (n = 8). (D) Schematic representation of experimental design; Nr4a1-GFP mice received treatment as shown in panel A and were analyzed with immunofluorescence following perfusion with PBS to wash out nonattached circulating blood cells. (E) Representative immunofluorescence images showing the uptake of DiI⁺ RBCs by GFP⁺ PMos on the endothelial surface of liver and lung in mice that received DiI-labeled sickle RBCs or control RBCs. CD31/CD144 (endothelial markers, blue), DiI (RBC in red), and GFP (PMo, green). Scale bar, 5 µm. (F) The frequencies of GFP⁺ PMos with uptake of DiI⁺ RBC (DiI⁺ GFP⁺ PMos) in mice as shown in panel E. (G) Representative IFC images acquired and gated on DiI⁺ PMos from hemin-treated Nr4a1-GFP reporter mice. Left to right: single-channel BF, GFP (representing PMos in green), DiI (representing sickle RBC material in red), CD45 (representing surface PMos in purple), and merged images showing DiI⁺ materials within GFP⁺CD45⁺ PMos. (H) Histogram depicting degree of DiI⁺ material internalized within GFP⁺ PMos with internalization score (<0 represents DiI⁺ material attached to the surface of PMo; >0 represents DiI⁺ material internalized by PMos). Percentage of cells with an internalization score >0 is indicated. Data represent mean ± SEM; means were compared using a 2-tailed Student t test. **P < .01; ***P < .001.