{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T10:12:56Z","timestamp":1792663976937,"version":"4.5.0"},"reference-count":34,"publisher":"Wiley","issue":"8","license":[{"start":{"date-parts":[[2024,8,14]],"date-time":"2024-08-14T00:00:00Z","timestamp":1723593600000},"content-version":"vor","delay-in-days":13,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["J of Extracellular Vesicle"],"published-print":{"date-parts":[[2024,8]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    High\u2010sensitivity flow cytometers have been developed for multi\u2010parameter characterization of single extracellular vesicles (EVs), but performance varies among instruments and calibration methods. Here we compare the characterization of identical (split) EV samples derived from human colorectal cancer (DiFi) cells by three high\u2010sensitivity flow cytometers, two commercial instruments, CytoFLEX\/CellStream, and a custom single\u2010molecule flow cytometer (SMFC). DiFi EVs were stained with the membrane dye di\u20108\u2010ANEPPS and with PE\u2010conjugated anti\u2010EGFR or anti\u2010tetraspanin (CD9\/CD63\/CD81) antibodies for estimation of EV size and surface protein copy numbers. The limits of detection (LODs) for immunofluorescence and vesicle size based on calibration using cross\u2010calibrated, hard\u2010dyed beads were \u223c10 PE\/\u223c80\u00a0nm EV diameter for CytoFLEX and \u223c10 PEs\/\u223c67\u00a0nm for CellStream. For the SMFC, the LOD for immunofluorescence was 1 PE and \u2264 35\u00a0nm for size. The population of EVs detected by each system (di\u20108\u2010ANEPPS\n                    <jats:sup>+<\/jats:sup>\n                    \/PE\n                    <jats:sup>+<\/jats:sup>\n                    particles) differed widely depending on the LOD of the system; for example, CellStream\/CytoFLEX detected only 5.7% and 1.5% of the tetraspanin\u2010labelled EVs detected by SMFC, respectively, and median EV diameter and antibody copy numbers were much larger for CellStream\/CytoFLEX than for SMFC as measured and validated using super\u2010resolution\/single\u2010molecule TIRF microscopy. To obtain a dataset representing a common EV population analysed by all three platforms, we filtered out SMFC and CellStream measurements for EVs below the CytoFLEX LODs as determined by bead calibration (10 PE\/80\u00a0nm). The inter\u2010platform agreement using this filtered dataset was significantly better than for the unfiltered dataset, but even better concordance between results was obtained by applying higher cutoffs (21 PE\/120\u00a0nm) determined by threshold analysis using the SMFC data. The results demonstrate the impact of specifying LODs to define the EV population analysed on inter\u2010instrument reproducibility in EV flow cytometry studies, and the utility of threshold analysis of SMFC data for providing semi\u2010quantitative LOD values for other flow cytometers.\n                  <\/jats:p>","DOI":"10.1002\/jev2.12498","type":"journal-article","created":{"date-parts":[[2024,8,14]],"date-time":"2024-08-14T07:44:46Z","timestamp":1723621486000},"source":"Crossref","is-referenced-by-count":38,"title":["Comparison of EV characterization by commercial high\u2010sensitivity flow cytometers and a custom single\u2010molecule flow cytometer"],"prefix":"10.1002","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6990-509X","authenticated-orcid":false,"given":"James","family":"Kim","sequence":"first","affiliation":[{"name":"Department of Chemistry University of Washington  Seattle Washington 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USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mengxia","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Chemistry University of Washington  Seattle Washington USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bryant S.","family":"Fujimoto","sequence":"additional","affiliation":[{"name":"Department of Chemistry University of Washington  Seattle Washington USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wyatt","family":"Nelson","sequence":"additional","affiliation":[{"name":"Department of Chemistry University of Washington  Seattle Washington USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Perry","family":"Schiro","sequence":"additional","affiliation":[{"name":"Micareo Inc  Burlingame California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeffrey L.","family":"Franklin","sequence":"additional","affiliation":[{"name":"Department of Medicine Vanderbilt University Medical Center  Nashville Tennessee USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"James N.","family":"Higginbotham","sequence":"additional","affiliation":[{"name":"Department of Medicine Vanderbilt University Medical Center  Nashville Tennessee USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2180-3844","authenticated-orcid":false,"given":"Robert J.","family":"Coffey","sequence":"additional","affiliation":[{"name":"Department of Medicine Vanderbilt University Medical Center  Nashville Tennessee USA"},{"name":"Department of Cell Biology Vanderbilt University  Nashville Tennessee USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Min","family":"Shi","sequence":"additional","affiliation":[{"name":"Department of Pathology University of Washington  Seattle Washington USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lucia 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