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Most analytical methods provide information on a particle mixture as a whole, without distinguishing viral particles from EVs. By utilising nano\u2010flow cytometry (nFC), a specialised form of flow cytometry adapted for the investigation of nanoparticles, we developed a simple, nucleic acid staining\u2010based method for discrimination and simultaneous quantification of the human cytomegalovirus (HCMV) virions, dense bodies and EVs, within extracellular particle mixtures produced by HCMV\u2010infected cells. We show that nucleic acid staining allows for discrimination of the individual particle types based on their distinct fluorescence\/side scatter profiles, assessed at single\u2010particle level by nFC. Following this, we optimised a method for physical separation of EVs from viral particles, based on high\u2010speed centrifugation through density cushions, using nFC as a tool to evaluate the purity of the isolated EVs. The methods introduced here have the capacity to circumvent common difficulties associated with the co\u2010investigation of EVs and viruses.<\/jats:p>","DOI":"10.1002\/jev2.70060","type":"journal-article","created":{"date-parts":[[2025,5,2]],"date-time":"2025-05-02T08:27:05Z","timestamp":1746174425000},"source":"Crossref","is-referenced-by-count":16,"title":["Nano\u2010Flow Cytometry\u2010Guided Discrimination and Separation of Human Cytomegalovirus Virions and Extracellular Vesicles"],"prefix":"10.1002","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5796-0799","authenticated-orcid":false,"given":"Vladimir","family":"Bokun","sequence":"first","affiliation":[{"name":"Institute of Reproductive and Developmental Biology, Department of Metabolism, Digestion and Reproduction, Faculty of Medicine Imperial College London  London UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Blair 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