Staining and Characterizing Extracellular Vesicles from Platelet-Poor Plasma Using the CytoFLEX nano Flow Cytometer 2306502

Abstract Introduction Extracellular vesicles (EVs) are recognized as important mediators of intercellular communication and potential diagnostic biomarkers. However, their precise characterization, particularly from complex biological samples like platelet-poor plasma (PPP), demands highly standardized methods. Our work establishes a complete process for the enrichment, fluorescent staining, and multicolor flow cytometric analysis of EVs from PPP using the CytoFLEX nano Flow Cytometer, leveraging its improved violet side scatter (VSSC-1) for sensitive detection. Methods Izon qEV SEC fractionation was used to enrich EVs from PPP, followed by serial dilutions of EV-enriched fractions to determine the optimal dilution at which the swarming effect is avoided. Thorough antibody titration was performed to minimize antibody aggregation and maintain background noise at the level set at the beginning of acquisition. Results During our investigation, we determined three crucial factors that could affect EV staining and thus their characterization: 1. Variation between different donors within the same fraction. 2. Differences among CD9 antibody clones in both the detected amount of PPP EVs and their Median Fluorescence Intensity (MFI), highlighting the importance of selecting the optimal clone for staining. 3. Confirmation of the importance of proper clone selection in multicolor analysis, along with demonstration of fluorochrome combinations using different fluorochromes conjugated to the same CD9 clone. In our multicolor flow cytometry analysis, we further showed that besides CD9, PPP EVs also express CD61, whereas CD63 and CD81 exhibited very low–if any–expression levels. Conclusion In conclusion, our standardized process demonstrates the importance of optimizing EV and sample preparation, along with careful attention to the EV fraction, donor variability, and antibody choices (clone, fluorochrome, and dilutions). These factors influence the accuracy and consistency of EV staining and characterization. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)