Fig. 3

Characterization of microglia-derived EVs in human plasma. (A) A representative. TEM image of EVs isolated from human plasma showed double-layered membrane-bound vesicles. (B) NTA showed a population of EVs with a peak around 70 nm. (C) Representative images of WB showed the expression of microglia-specific markers (CX3CR1 and UCHL1) and EV markers (ALIX and CD9) in the isolated EVs from plasma, raw human plasma and the supernatant of plasma ultracentrifugation. (D) Typical STORM images showed CX3CR1 and UCHL1 colocalized on the CD9 marked EV membranes. Moreover, NFL was localized on the CD9-marked EV membranes. Notably, the size of EV signals in STORM exceeds 200 nm, which may be attributed to PFA fixation and the algorithm-based approach, both of which introduce certain biases when characterizing the size of EVs. (E) Representative histograms showed the populations of EVs positive for each marker (CX3CR1, UCHL1, and NFL), fluorophore-conjugated IgG isotype control, a blank (fluorophore only, no antibody) control experiment, and UC-depleted (plasma with depletion of EVs by ultracentrifugation). Quantification data of positive EVs detected by the flow cytometry-based assay demonstrating the specificity of EV assays. (F) Representative images for double labeling of microglial EV markers (CX3CR1 and UCHL1) and isotype IgG in a pooled human plasma sample (combined plasma from 40 individuals). All values are mean ± SEM. n = 3 in each group