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Tumor\u2010derived extracellular vesicles (TD\u2010EVs) are key players in this process. They can interact with immune cells and modulate their activity, either suppressing or activating the immune system. Deciphering the interactions between TD\u2010EVs and immune cells is essential to understand immune modulation by cancer cells. Fluorescent labelling of TD\u2010EVs is a method of choice to study such interaction. This work aims to determine the impact of EV labelling methods on the detection by imaging flow cytometry and multicolour spectral flow cytometry of EV interaction and capture by the different immune cell types within human Peripheral Blood Mononuclear Cells (PBMCs). EVs released by the triple\u2010negative breast carcinoma cell line MDA\u2010MB\u2010231 were labelled either with the lipophilic dye MemGlow\u2010488 (MG\u2010488), Carboxyfluorescein diacetate, succinimidyl ester (CFDA\u2010SE) or through ectopic expression of a MyrPalm\u2010superFolderGFP reporter (mp\u2010sfGFP), which incorporates into EVs during their biogenesis. Our results show that these labelling strategies, although analysed with the same techniques, led to diverging results. While MG\u2010488\u2010labelled EVs incorporate in all cell types, CFSE\u2010labelled EVs are restricted to a minor subset of cells and mp\u2010sfGFP\u2010labelled EVs are mainly detected in CD14+ monocytes which are the main uptakers of EVs and other particles, regardless of the labelling method. Furthermore, our results show that the method used for EV labelling influences the detection of the different types of EV interactions with the recipient cells. Specifically, MG\u2010488, CFSE and mp\u2010sfGFP result in observation suggesting, respectively, transient EV\u2010PM interaction that results in dye transfer, EV content delivery, and capture of intact EVs. Consequently, the type of EV labelling method has to be considered as they can provide complementary information on various types of EV\u2010cell interaction and EV fate.<\/jats:p>","DOI":"10.1002\/jev2.12384","type":"journal-article","created":{"date-parts":[[2023,11,30]],"date-time":"2023-11-30T05:58:31Z","timestamp":1701323911000},"source":"Crossref","is-referenced-by-count":45,"title":["Detection of the interactions of tumour derived extracellular vesicles with immune cells is dependent on EV\u2010labelling methods"],"prefix":"10.1002","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-2165-6211","authenticated-orcid":false,"given":"Luisa","family":"Loconte","sequence":"first","affiliation":[{"name":"Institut Curie, PSL University Inserm U932 Immunity and Cancer  Paris France"},{"name":"Istituto Pasteur Italia\u2010Fondazione Cenci Bolognetti, Department of Molecular Medicine Sapienza\u2019 University of Rome  Rome 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