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. 2022 Apr 27;13:853769. doi: 10.3389/fimmu.2022.853769

Figure 2.

Figure 2

In vivo uptake of MVs. (A) Flow cytometry plot of RAW macrophage derived MVs, demonstrating that DiD labeled events were also positive for CD11b, confirming their identity as macrophage-derived MVs. (B) These particles were readily visualized by confocal microscopy as DiD (red, top left panel), particles negative for nuclear materials (DAPI-, top-right), but positive for CD11b (green, bottom-left, co-localization shown in the bottom-right combined image) (n = 3). (C) DiD labeled RAW macrophage MVs (25,000 RFU) were instilled into the trachea of untreated mice and in vivo MV uptake by alveolar macrophages and epithelial cells was assessed by flow cytometry at 1 and 4 hours. Time 0 represents baseline auto-fluorescence, i.e. no MVs added. Alveolar macrophages significantly internalized the majority of MVs compared to alveolar epithelial cells at both time points [1 hour: alveolar macrophage 734 ± 135 RFU vs. epithelial cells 4.78 ± 0.57 RFU; 4 hours: alveolar macrophage 1153 ± 243 RFU vs. epithelial cells 5.21 ± 0.47 RFU (n = 5)]. (D) Alveolar macrophages, as compared to epithelial cells, internalized the majority of MVs at 1 hour after installation in vivo regardless of the MV phenotypes used: 1) in vitro-derived RAW macrophage MVs (i.e. from stimulated RAW macrophage culture); 2) in vivo-derived MVs, non-inflamed (intra-alveolar MVs harvested by lung lavage from untreated mice); and 3) in vivo-derived MVs, inflamed (intra-alveolar MVs harvested by lung lavage from LPS-treated mice). (n = 3-6, not significant by one way ANOVA).