FIGURE 2.

Uptake of RBCEVs by macrophages is mediated by phosphatidylserine. (a) Schematic of the experimental strategy and flow cytometry analysis data showing RBCEV uptake by PBMC‐derived macrophages after pre‐incubation with phosphatidylserine liposomes (PS‐lipo) or phosphatidylcholine liposomes (PC‐lipo) at different concentrations for 30 min. The cells were then incubated with CFSE‐labelled RBCEVs for 2 h. (b) Schematic of the experimental strategy and data from nanoparticle flow cytometry analysis showing Annexin V staining of phosphatidylserine (PS) on the surface of RBCEVs. CFSE‐labelled RBCEVs were treated with methyl‐α‐cyclodextrin and 1,2‐distearoyl‐sn‐glycero‐3‐phosphocholine (DSPC) to reduce the expression of PS on their outer leaflet membrane (PS‐reduced). L‐α‐phosphatidylserine was added to the PS‐reduced EVs to restore PS expression (PS‐restored). Untreated RBCEVs and modified RBCEVs were stained with Annexin V for PS detection. The upper row of contour plots shows the controls and gating strategy for the CFSE‐positive population. Subsequently, Annexin V signals were gated based on the CFSE‐positive particles (n = 4). (c) Flow cytometry analysis of CFSE signals indicating the uptake of RBCEVs treated as described in (b) by macrophages. After PS removal or PS restoration, RBCEVs were incubated with macrophages for 2 h (n = 8). All the bar graphs represent the mean ± SD. Student's two‐tailed t‐test (b,c), **p < 0.01, *p < 0.05, ns: not significant (p > 0.05). a.u.: arbitrary unit.