FIGURE 1.

RBCEVs are taken up preferentially by macrophages and monocytes. (a) Schematic of the experimental setup used to track the biodistribution of RBCEVs. 500 μg of Aco‐490‐labelled RBCEVs were injected intravenously into C57BL/6 mice. The organs were collected 8 h later for analysis. (b) Flow cytometry analysis of Aco‐490 signals in dissociated liver, spleen, lung, and bone marrow as outlined in (a). Data are presented as the percentage of Aco‐490‐positive cells. (c) Confocal images of the liver and spleen sections as outlined in (a). Macrophages were identified by immunolabelling with F4/80 or CD169 antibodies (Red). Nuclei were stained with NucSpot® Live 488 (Cyan). Scale bars, 100 μm. (d) Flow cytometry analysis of Aco‐490 signals in PBMCs incubated with Aco‐490‐labelled RBCEVs for 2 h or 24 h and stained with antibodies for the surface markers to identify T‐cells, NK cells, monocytes, and B cells. (e) Quantification of CFSE‐labelled RBCEVs taken up by different cell types after incubation with 40 μg of RBCEVs for 2 h. (f) Flow cytometry analysis quantifying RBCEV uptake by macrophages differentiated from human CD14+ PBMCs in M‐CSF for 6 d. Macrophages were either unstimulated (M0), stimulated with LPS and IFN‐γ for 1 d, or stimulated with IL4 and IL10 for 1 d before incubation with CFSE‐labelled RBCEVs for 2 h. All the bar graphs represent the mean ± SD. a.u.: arbitrary unit. Student's two‐tailed t‐test (f), **p < 0.01, *p < 0.05, ns: not significant (p > 0.05).