Growing interest in extracellular vesicles (EVs) has prompted the advancements of protocols for improved EV characterization. As a high-throughput, multi-parameter, and single particle technique, flow cytometry is widely used for EV characterization. The comparison of data on EV concentration, however, is hindered by the lack of standardization between different protocols and instruments. Here, we quantified EV counts of platelet-derived EVs, using two flow cytometers (Gallios and CytoFLEX LX) and nanoparticle tracking analysis (NTA). Phosphatidylserine-exposing EVs were identified by labelling with lactadherin (LA). Calibration with silica-based fluorescent beads showed detection limits of 300 nm and 150 nm for Gallios and CytoFLEX LX, respectively. Accordingly, CytoFLEX LX yielded 40-fold higher EV counts and 13-fold higher counts of LA+CD41+ EVs compared to Gallios. NTA in fluorescence mode (F-NTA) demonstrated that only 9.5% of all vesicles detected in scatter mode exposed phosphatidylserine, resulting in good agreement of LA+ EVs for CytoFLEX LX and F-NTA. Since certain functional characteristics, such as the exposure of pro-coagulant phosphatidylserine, are not equally displayed across the entire EV size range, our study highlights the necessity of indicating the size range of EVs detected with a given approach along with the EV concentration to support the comparability between different studies.
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