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Vault particle components have been repeatedly reported in the literature as EV cargo. Here, we demonstrated that vault RNA (vtRNA) and major vault protein (MVP) were highly abundant in EV pellets enriched by differential centrifugation (DC) by qPCR and western blotting, respectively. RNase and proteinase treatment of DC\u2010derived pellets demonstrated that most vtRNA and MVP were not enclosed and protected within an EV membrane. Vault\u2010like particles were visualised in 100k DC pellets by cryo\u2010transmission electron microscopy. EVs were enriched by size exclusion chromatography, and western blotting of individual fractions showed co\u2010elution of EV markers and vault particle proteins. Immunocapture of EVs post\u2010ultracentrifugation (100k DC pellet) showed co\u2010purification of MVP, whereas EVs isolated by direct immunocapture from conditioned medium were MVP\u2010negative. The current study highlights the importance of determining the topology of putative EV\u2010associated components to determine if they are EV cargo or contaminants that have been co\u2010purified.<\/jats:p>","DOI":"10.1002\/jev2.70142","type":"journal-article","created":{"date-parts":[[2025,8,6]],"date-time":"2025-08-06T09:55:31Z","timestamp":1754474131000},"source":"Crossref","is-referenced-by-count":6,"title":["Assessment of Vault Particles in Cancer Cell Line\u2010Derived Extracellular Vesicle Preparations"],"prefix":"10.1002","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8809-3907","authenticated-orcid":false,"given":"Xinming","family":"Liu","sequence":"first","affiliation":[{"name":"School of Clinical Dentistry The University of Sheffield  Sheffield UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5552-3656","authenticated-orcid":false,"given":"Zubair 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