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Here we introduce free\u2010flow electrophoresis (FFE) as a novel approach for purifying EVs from primary biofluids, in particular from the peritoneal fluid (ascites) of ovarian cancer patients. FFE represents a versatile, fast, matrix\u2010free approach for separating different analytes with inherent differences in charge density and\/or isoelectric point (pI). Using a series of buffered media with different pH values allowed us to collect 96 fractions of ascites samples. To characterize the composition of the individual fractions, we used state\u2010of\u2010the\u2010art methods such as nanoflow and imaging flow cytometry (nFCM and iFCM) in addition to classical approaches. Of note, tetraspanin\u2010positive events measured using nFCM were enriched in a small number of distinct fractions. This observation was corroborated by Western blot analysis and electron microscopy, demonstrating only minor contamination with soluble proteins and lipid particles. In addition, these gently purified EVs remain functional. Thus, FFE represents a new, efficient and fast method for separating native and highly purified EVs from complicated primary samples.<\/jats:p>","DOI":"10.1002\/jex2.71","type":"journal-article","created":{"date-parts":[[2022,12,26]],"date-time":"2022-12-26T02:31:50Z","timestamp":1672021910000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Isolation of native EVs from primary biofluids\u2014Free\u2010flow electrophoresis as a novel approach to purify ascites\u2010derived EVs"],"prefix":"10.1002","volume":"1","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5183-4262","authenticated-orcid":false,"given":"Christian","family":"Preu\u00dfer","sequence":"first","affiliation":[{"name":"Institute for Tumor Immunology, Center for Tumor Biology and Immunology Philipps University Marburg  Marburg Germany"},{"name":"Core Facility Extracellular Vesicles, Center for Tumor Biology and Immunology Philipps University of Marburg  Marburg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kathrin","family":"Stelter","sequence":"additional","affiliation":[{"name":"Institute for Tumor Immunology, Center for Tumor Biology and Immunology Philipps University Marburg  Marburg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tobias","family":"Tertel","sequence":"additional","affiliation":[{"name":"Institute for Transfusion Medicine University Hospital Essen University of Duisburg\u2010Essen  Essen Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Manuel","family":"Linder","sequence":"additional","affiliation":[{"name":"Institute for Tumor Immunology, Center for Tumor Biology and Immunology Philipps University Marburg  Marburg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Frederik","family":"Helmprobst","sequence":"additional","affiliation":[{"name":"Core Facility for Mouse Pathology and Electron Microscopy, Institute of Neuropathology Philipps University Marburg  Marburg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Witold","family":"Szymanski","sequence":"additional","affiliation":[{"name":"Institute of Translational Proteomics Philipps University of Marburg  Marburg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3015-5850","authenticated-orcid":false,"given":"Johannes","family":"Graumann","sequence":"additional","affiliation":[{"name":"Institute of Translational Proteomics Philipps University of Marburg  Marburg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bernd","family":"Giebel","sequence":"additional","affiliation":[{"name":"Institute for Transfusion Medicine University Hospital Essen University of Duisburg\u2010Essen  Essen Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Silke","family":"Reinartz","sequence":"additional","affiliation":[{"name":"Translational Oncology Group, Center for Tumor Biology and Immunology Philipps University Marburg  Marburg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rolf","family":"M\u00fcller","sequence":"additional","affiliation":[{"name":"Translational Oncology Group, Center for Tumor Biology and Immunology Philipps University Marburg  Marburg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gerhard","family":"Weber","sequence":"additional","affiliation":[{"name":"FFE Service GmbH  Feldkirchen Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4785-9165","authenticated-orcid":false,"given":"Elke Pogge","family":"von Strandmann","sequence":"additional","affiliation":[{"name":"Institute for Tumor Immunology, Center for Tumor Biology and Immunology Philipps University Marburg  Marburg Germany"},{"name":"Core Facility Extracellular Vesicles, Center for Tumor Biology and Immunology Philipps University of Marburg  Marburg 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