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The isolation of EPs from supernatants, biofluids, and solid tissues allows further research into understanding the role of EPs in physiological and pathological scenarios, which aids in the development of EP-based therapies in biomedicine. This article presents a straightforward, direct, and applicable method for isolating and characterizing EPs from three regions of the porcine brain: the cerebrum, cerebellum, and brainstem. The protocol is a method based on three steps: enzymatic treatment, differential centrifugation, and filtration\/ultrafiltration to isolate the brain\u2019s EPs. Analysis by scanning electron microscopy (SEM) and nanoparticle tracking analysis (ZetaView) revealed the enrichment of the brain\u2019s EPs in the size range of 20\u2013200\u2009nm when isolated using this protocol. Additionally, CD63 and HSP70 expression was assessed by Western Blot without finding significant differences between the brain regions. This simple adapted method will help the understanding of extracellular ecosystems in the CNS and could have interesting implications in brain diagnosis and therapy.<\/jats:p>","DOI":"10.1371\/journal.pone.0329985","type":"journal-article","created":{"date-parts":[[2025,8,27]],"date-time":"2025-08-27T17:26:58Z","timestamp":1756315618000},"page":"e0329985","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["Protocol for the isolation and characterization of porcine brain region-associated extracellular particles"],"prefix":"10.1371","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5436-3558","authenticated-orcid":true,"given":"Abigail","family":"De Avila","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mayra","family":"Diaz","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bruno 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