An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 45 Tetraspanin distribution on human plasma-derived single EV After having established that our IFCM methodology identifies and quantifies single EV through staining with CFDA-SE and the anti-tetraspanin antibody mixture, we aimed to analyze whether we could detect different subsets of EV. Therefore, we assessed the contributions of the individual tetraspanins to the double-positive events pool. The 5 PPP samples were stained with CFDA-SE and either the antitetraspanin antibody mixture or one of its individual components (anti-CD9 [clone HI9a], anti-CD63 [clone H5C5] or anti-CD81 [clone 5A6]) at a concentration equal to that used within the mixture. The concentrations of double-positive events upon staining with each stain were compared (Figure 6a) and normalized with respect to the concentration of double-positive events (in objects/mL) obtained with the anti-tetraspanin antibody mixture (Figure 6b). The tetraspanin marker CD9 was found to be the main contributor to the fluorescent signal and thus responsible for most of the double-positive EV identified in PPP when stained with the anti-tetraspanin antibody mixture: ~88 ± 11% of the doublepositive events were still present when staining with only CD9 versus ~13 ± 3% for CD63 and ~9 ± 5% for CD81. In short, we show that our methodology is able to identify subsets of EV, and that tetraspanin marker CD9 – and not CD63 or CD81 - represent the bulk of CFSE+ single EV in PPP of healthy individuals.
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