{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T04:23:26Z","timestamp":1791433406788,"version":"4.3.3"},"reference-count":27,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2019,12,31]],"date-time":"2019-12-31T00:00:00Z","timestamp":1577750400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100008271","name":"LEO Pharma Research Foundation","doi-asserted-by":"publisher","award":["180208"],"award-info":[{"award-number":["180208"]}],"id":[{"id":"10.13039\/501100008271","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Anelise\u2010Asmussen foundation","award":["180802"],"award-info":[{"award-number":["180802"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The field of extracellular vesicle (EV) research is challenged by the lack of standardized protocols to identify and specifically distinguish between exosomes and ectosomes, which are released via exocytosis or plasma membrane shedding, respectively. Using sequential centrifugation, we separated EV subpopulations from supernatants of COLO 357 pancreas carcinoma cells based on size and mass. After 10,000\u00d7 g centrifugation, we reconstituted high-speed (hs) EVs from the pellet, directly labeled them with the membrane dye carboxyfluorescein diacetate succinimidyl ester (CFSE), and performed flow cytometry based analysis. The aim was to optimize the conditions for EV labeling and detection and hence to obtain a maximum yield of intact hsEVs. We found that, for sufficient labeling of EVs, minimal temperature variations and short incubation times correlated with EV stability. Furthermore, threshold adjustment significantly improved the sensitivity of the flow cytometer for the detection of CFSE labeled hsEVs. When cells were CFSE labeled, we observed a transition of fluorescence onto EVs that were reconstituted from the pellet but not onto those that remained in the supernatant after hs centrifugation, suggesting the indirect labeling of EVs based on the way of biogenesis as a specific method for the distinction of exosomes and ectosomes. Protocol standardization is of major importance for the use of EVs as diagnostic markers in liquid biopsies.<\/jats:p>","DOI":"10.3390\/ijms21010291","type":"journal-article","created":{"date-parts":[[2020,1,3]],"date-time":"2020-01-03T04:43:03Z","timestamp":1578026583000},"page":"291","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":44,"title":["Detection and Quantification of Extracellular Vesicles via FACS: Membrane Labeling Matters!"],"prefix":"10.3390","volume":"21","author":[{"given":"Fanny","family":"Ender","sequence":"first","affiliation":[{"name":"Experimental Oncology, University Hospital and Medical School (UKSH), University of Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Piet","family":"Zamzow","sequence":"additional","affiliation":[{"name":"Experimental Oncology, University Hospital and Medical School (UKSH), University of Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nikolas","family":"von Bubnoff","sequence":"additional","affiliation":[{"name":"Experimental Oncology, University Hospital and Medical School (UKSH), University of Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6409-3822","authenticated-orcid":false,"given":"Frank","family":"Gieseler","sequence":"additional","affiliation":[{"name":"Experimental Oncology, University Hospital and Medical School (UKSH), University of Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2019,12,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"3395","DOI":"10.21873\/anticanres.13483","article-title":"Extracellular vesicles: Subcellular organelles with the potential to spread cancer resistance","volume":"39","author":"Ender","year":"2019","journal-title":"Anticancer Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"27066","DOI":"10.3402\/jev.v4.27066","article-title":"Biological properties of extracellular vesicles and their physiological functions","volume":"4","author":"Siljander","year":"2015","journal-title":"J. Extracell. Vesicles"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"364","DOI":"10.1016\/j.tcb.2015.01.004","article-title":"Ectosomes and exosomes: Shedding the confusion between extracellular vesicles","volume":"25","author":"Cocucci","year":"2015","journal-title":"Trends Cell Boil."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1535750","DOI":"10.1080\/20013078.2018.1535750","article-title":"Minimal information for studies of extracellular vesicles 2018 (MISEV2018): A position statement of the international society for extracellular vesicles and update of the MISEV2014 guidelines","volume":"7","author":"Witwer","year":"2018","journal-title":"J. Extracell. Vesicles"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"27066","DOI":"10.3402\/jev.v5.32945","article-title":"Techniques used for the isolation and characterization of extracellular vesicles: Results of a worldwide survey","volume":"5","author":"Gardiner","year":"2016","journal-title":"J. Extracell. Vesicles"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"20677","DOI":"10.3402\/jev.v2i0.20677","article-title":"Distinct RNA profiles in subpopulations of extracellular vesicles: Apoptotic bodies, microvesicles and exosomes","volume":"2","author":"Crescitelli","year":"2013","journal-title":"J. Extracell. Vesicles"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1050","DOI":"10.1002\/cbin.10645","article-title":"Extracellular vesicles from malignant effusions induce tumor cell migration: Inhibitory effect of LMWH tinzaparin","volume":"40","author":"Gamperl","year":"2016","journal-title":"Cell Boil. Int."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"78","DOI":"10.3389\/fcell.2017.00078","article-title":"Extracellular vesicle flow cytometry analysis and standardization","volume":"5","author":"Welsh","year":"2017","journal-title":"Front. Cell Dev. Boil."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1002\/0471142735.im0409s84","article-title":"Use of the intracellular fluorescent dye cfse to monitor lymphocyte migration and proliferation","volume":"84","author":"Parish","year":"2009","journal-title":"Curr. Protoc. Immunol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1533","DOI":"10.1002\/cbin.11040","article-title":"Heterogeneity of microvesicles from cancer cell lines under inflammatory stimulation with TNF-\u03b1","volume":"42","author":"Gieseler","year":"2018","journal-title":"Cell Boil. Int."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"e1700716","DOI":"10.1002\/biot.201700716","article-title":"Extracellular vesicles in oncology: Progress and pitfalls in the methods of isolation and analysis","volume":"14","author":"Lucchetti","year":"2019","journal-title":"Biotechnol. J."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2571","DOI":"10.1111\/j.1538-7836.2010.04047.x","article-title":"Standardization of platelet-derived microparticle enumeration by flow cytometry with calibrated beads: Results of the international society on thrombosis and haemostasis SSC collaborative workshop","volume":"8","author":"Lacroix","year":"2010","journal-title":"J. Thromb. Haemost."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1875","DOI":"10.1016\/j.cub.2009.09.059","article-title":"ARF6-regulated shedding of tumor cell-derived plasma membrane microvesicles","volume":"19","author":"Clancy","year":"2009","journal-title":"Curr. Boil."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1185","DOI":"10.1160\/TH11-07-0509","article-title":"Correlation between platelet-derived microparticle enumeration by flow cytometry and phospholipid-dependent procoagulant activity in microparticles: The centrifugation step matters!","volume":"107","author":"Stagnara","year":"2012","journal-title":"Thromb. Haemost."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"407","DOI":"10.1146\/annurev.biophys.093008.131234","article-title":"The distribution and function of phosphatidylserine in cellular membranes","volume":"39","author":"Leventis","year":"2010","journal-title":"Annu. Rev. Biophys."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1038\/nrm.2017.125","article-title":"Shedding light on the cell biology of extracellular vesicles","volume":"19","author":"Raposo","year":"2018","journal-title":"Nat. Rev. Mol. Cell Boil."},{"key":"ref_17","unstructured":"Shapiro, H.M. (2005). Practical Flow Cytometry, John Wiley & Sons. [4th ed.]."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1002\/cyto.a.20134","article-title":"Refractive index measurement in viable cells using quantitative phase-amplitude microscopy and confocal microscopy","volume":"65","author":"Curl","year":"2005","journal-title":"Cytom. Part A"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Szatanek, R., Baj-Krzyworzeka, M., Zimoch, J., Lekka, M., Siedlar, M., and Baran, J. (2017). The methods of choice for Extracellular Vesicles (EVs) characterization. Int. J. Mol. Sci., 18.","DOI":"10.3390\/ijms18061153"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"447","DOI":"10.1021\/bc00034a015","article-title":"Comparison of three common amine reactive fluorescent probes used for conjugation to biomolecules by capillary zone electrophoresis","volume":"6","author":"Banks","year":"1995","journal-title":"Bioconjugate Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"499","DOI":"10.1046\/j.1440-1711.1999.00877.x","article-title":"Fluorescent dyes for lymphocyte migration and proliferation studies","volume":"77","author":"Parish","year":"1999","journal-title":"Immunol. Cell Boil."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1016\/0022-1759(90)90322-M","article-title":"New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy","volume":"133","author":"Weston","year":"1990","journal-title":"J. Immunol. Methods"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1878","DOI":"10.1038\/s41598-017-01731-2","article-title":"Labeling extracellular vesicles for nanoscale flow cytometry","volume":"7","author":"Telford","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"5.3.1","DOI":"10.1002\/0471142735.im0503s44","article-title":"Preparation of cells and reagents for flow cytometry","volume":"44","author":"Holmes","year":"2001","journal-title":"Curr. Protoc. Immunol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"680","DOI":"10.1373\/clinchem.2017.278978","article-title":"Comparison of generic fluorescent markers for detection of extracellular vesicles by flow cytometry","volume":"64","author":"Hau","year":"2018","journal-title":"Clin. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1002\/cyto.a.22669","article-title":"Fluorescence triggering: A general strategy for enumerating and phenotyping extracellular vesicles by flow cytometry","volume":"89","author":"Arraud","year":"2016","journal-title":"Cytometry A"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1002\/ijc.2910250507","article-title":"Human cell line (COLO 357) of metastatic pancreatic adenocarcinoma","volume":"25","author":"Morgan","year":"1980","journal-title":"Int. J. Cancer"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/1\/291\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T13:47:20Z","timestamp":1760190440000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/1\/291"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,12,31]]},"references-count":27,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2020,1]]}},"alternative-id":["ijms21010291"],"URL":"https:\/\/doi.org\/10.3390\/ijms21010291","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,12,31]]}}}