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In this study, a laboratory\u2010built nano\u2010flow cytometer (nFCM) that can detect single EVs as small as 40\u00a0nm in diameter and single DNA fragments of 200\u00a0bp upon SYTO 16 staining was used to study EV\u2010DNA at the single\u2010vesicle level. Through simultaneous side\u2010scatter and fluorescence (FL) detection of single particles and with the combination of enzymatic treatment, present study revealed that: (1) naked DNA or DNA associated with non\u2010vesicular entities is abundantly presented in EV samples prepared from cell culture medium by ultracentrifugation; (2) the quantity of EV\u2010DNA in individual EVs exhibits large heterogeneity and the population of DNA positive (DNA<jats:sup>+<\/jats:sup>) EVs varies from 30% to 80% depending on the cell type; (3) external EV\u2010DNA is mainly localized on relatively small size EVs (e.g. &lt;100 nm for HCT\u201015 cell line) and the secretion of external DNA<jats:sup>+<\/jats:sup> EVs can be significantly reduced by exosome secretion pathway inhibition; (4) internal EV\u2010DNA is mainly packaged inside the lumen of relatively large EVs (e.g. 80\u2013200\u00a0nm for HCT\u201015 cell line); (5) double\u2010stranded DNA (dsDNA) is the predominant form of both the external and internal EV\u2010DNA; (6) histones (H3) are not found in EVs, and EV\u2010DNA is not associated with histone proteins and (7) genotoxic drug induces an enhanced release of DNA<jats:sup>+<\/jats:sup> EVs, and the number of both external DNA<jats:sup>+<\/jats:sup> EVs and internal DNA<jats:sup>+<\/jats:sup> EVs as well as the DNA content in single EVs increase significantly. This study provides direct and conclusive experimental evidence for an in\u2010depth understanding of how DNA is associated with EVs.<\/jats:p>","DOI":"10.1002\/jev2.12206","type":"journal-article","created":{"date-parts":[[2022,4,4]],"date-time":"2022-04-04T04:19:50Z","timestamp":1649045990000},"source":"Crossref","is-referenced-by-count":194,"title":["Analysis of extracellular vesicle DNA at the single\u2010vesicle level by nano\u2010flow cytometry"],"prefix":"10.1002","volume":"11","author":[{"given":"Haisheng","family":"Liu","sequence":"first","affiliation":[{"name":"Department of Chemical Biology MOE Key Laboratory of Spectrochemical Analysis &amp; Instrumentation Key Laboratory for Chemical Biology of Fujian Province Collaborative Innovation Center of Chemistry for Energy Materials College of Chemistry and Chemical Engineering Xiamen University  Xiamen People's Republic of 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