Fig. 1.

Extracellular vesicle (EV) isolation and characterization. A, Glioma cells release small EVs from multivesicular bodies (MVB), coordinated by the endosomal sorting complex required for transport (ESCRT), while larger EVs are generated by membrane budding. Glioma tissue was cultured and EVs secreted by tumor cells were analyzed. B, Electron microscopy demonstrates the cup-shaped morphology of EVs (arrows). C, Detection of CD9, CD63, and CD81 by imaging flow cytometry. D, Direct stochastic optical reconstruction microscopy of EVs identified by CD63 or CD81 and stained with nucleic acid dye. DNase treatment removed extra-vesicular DNA, and permeabilization accessed intra-vesicular DNA. E, Quantification of the number of DNA fragment localizations per EV, based on co-localization with CD63 and/or CD81. Box plots with 10-90 percentile (whiskers), median (line) and 25-75 percentile (box); ****P < .0001, Kruskal-Wallis analysis.