{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,19]],"date-time":"2026-10-19T22:36:41Z","timestamp":1792449401140,"version":"4.5.0"},"reference-count":31,"publisher":"Wiley","issue":"5","license":[{"start":{"date-parts":[[2025,5,19]],"date-time":"2025-05-19T00:00:00Z","timestamp":1747612800000},"content-version":"vor","delay-in-days":18,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000052","name":"NIH Office of the Director","doi-asserted-by":"publisher","award":["R03OD028265"],"award-info":[{"award-number":["R03OD028265"]}],"id":[{"id":"10.13039\/100000052","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100005508","name":"Boettcher Foundation","doi-asserted-by":"publisher","award":["Webb Waring Biomedical Research Award"],"award-info":[{"award-number":["Webb Waring Biomedical Research Award"]}],"id":[{"id":"10.13039\/100005508","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000054","name":"National Cancer Institute","doi-asserted-by":"publisher","award":["P30 CA046934"],"award-info":[{"award-number":["P30 CA046934"]}],"id":[{"id":"10.13039\/100000054","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["T32GM136628"],"award-info":[{"award-number":["T32GM136628"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000098","name":"Clinical Center","doi-asserted-by":"publisher","award":["L30 TR002126"],"award-info":[{"award-number":["L30 TR002126"]}],"id":[{"id":"10.13039\/100000098","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["J of Extracellular Vesicle"],"published-print":{"date-parts":[[2025,5]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>Current approaches for the fluorescent labelling of extracellular vesicles (EVs) have been reported to produce widely variable and controversial results, highlighting a significant need for validated, reproducible labelling methods to advance the field of EV research. Lipophilic membrane dyes are commonly used but have been shown to produce non\u2010specific fluorescent particles that are indistinguishable from labelled EVs, confounding experimental results. We aimed to distinguish conditions that can either promote or reduce the formation of non\u2010specific dye particles when using the prototypical lipophilic membrane dye PKH26. We optimised a labelling approach that minimises the production of non\u2010specific dye particles by altering buffer conditions during staining and validated this method across cell\u2010based and\u00a0in vivo systems of EV biodistribution. To do this, we specifically isolated small EVs using ultrafiltration and size exclusion chromatography and validated sample purity and post\u2010isolation processing steps. We then used single\u2010EV spectral flow cytometry and transmission electron microscopy to investigate the impact of four different buffer conditions on PKH26 non\u2010specific particle formation. We also determined the extent to which non\u2010specific PKH26 particles were detectable in cell\u2010based assays and in vivo within mouse lymph nodes using flow cytometry, immunofluorescence, and intravital imaging. By optimising buffer conditions to eliminate additional protein, we were able to minimise the formation of dye aggregates while maintaining efficient EV labelling, producing a much higher signal\u2010to\u2010noise ratio both in vitro and in vivo. We also demonstrate that failure to include proper vehicle controls can have significant implications on experimental results, leading to false positive data. This work emphasizes the importance of adequately benchmarking EV labelling approaches as it is essential for accurate evaluation of EV trafficking in physiologic and pathologic states.<\/jats:p>","DOI":"10.1002\/jev2.70079","type":"journal-article","created":{"date-parts":[[2025,5,19]],"date-time":"2025-05-19T10:12:18Z","timestamp":1747649538000},"source":"Crossref","is-referenced-by-count":23,"title":["Non\u2010Specific Particle Formation During Extracellular Vesicle Labelling With the Lipophilic Membrane Dye PKH26"],"prefix":"10.1002","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8410-248X","authenticated-orcid":false,"given":"Laurel A.","family":"Haines","sequence":"first","affiliation":[{"name":"Department of Microbiology, Immunology, &amp; Pathology College of Veterinary and Biomedical Sciences Colorado State University  Fort Collins Colorado USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4348-6325","authenticated-orcid":false,"given":"Alex A.","family":"Baeckler","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Immunology, &amp; Pathology College of Veterinary and Biomedical Sciences Colorado State University  Fort Collins Colorado USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sophi J.","family":"Schofield","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Immunology, &amp; Pathology College of Veterinary and Biomedical Sciences Colorado State University  Fort Collins Colorado USA"},{"name":"Department of Clinical Sciences College of Veterinary Medicine North Carolina State University  Raleigh North Carolina USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eric P.","family":"Palmer","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Immunology, &amp; Pathology College of Veterinary and Biomedical Sciences Colorado State University  Fort Collins Colorado USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bradley F.","family":"Guilliams","sequence":"additional","affiliation":[{"name":"Department of Chemistry College of Natural Sciences Colorado State University  Fort Collins Colorado USA"},{"name":"Analytical Resources Core Center for Imaging and Surface Science Colorado State University  Fort Collins Colorado USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Melinda A.","family":"Meyers","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences College of Veterinary and Biomedical Sciences, Colorado State University  Fort Collins Colorado USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel P.","family":"Regan","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Immunology, &amp; Pathology College of Veterinary and Biomedical Sciences Colorado State University  Fort Collins Colorado USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,5,19]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13287-024-04005-7"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.7150\/thno.17092"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1093\/biomet\/93.3.491"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1002\/jev2.12351"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1038\/ncb3169"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/1869"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-020-66434-7"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0138849"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1002\/JLB.1MIA0321-164R"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1093\/brain\/awac261"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00018-018-2773-4"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.3390\/cancers13174435"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2104826118"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1038\/srep17319"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00432-023-04682-6"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-021-04225-4"},{"key":"e_1_2_9_18_1","unstructured":"National Center for Biotechnology Information.1988.National Library of Medicine National Center for Biotechnology Information.https:\/\/www.ncbi.nlm.nih.gov\/. 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