Evaluation of Vesiculab Vesi-Ref CD63- mNeonGreen (mNG) Extracellular Vesicles (EV) Reference Standard on the CytoFLEX nano Flow Cytometer 2306506

Abstract

Abstract Introduction Accurate and reproducible characterization of EVs by nanoscale flow cytometry requires both robust reference materials and carefully controlled experimental conditions. This study evaluated the performance of the Vesiculab Vesi-Ref CD63-mNG EV reference standard using the Beckman Coulter Life Sciences CytoFLEX nano Flow Cytometer. Methods Our experimental design involved determining the optimal dilution of unstained Vesi-Ref CD63-mNG EVs through serial dilution in 20-nm-filtered PBS. This step aimed to achieve optimal target event rates (1000—2000 events per second) and minimize swarming by monitoring VSSC1-H scatter profiles. Concurrently, CD63-Alexa Fluor 647 antibody titrations (0.25-2.0 µg/mL) were performed to assess antibody aggregation and background signal. Subsequently, stained Vesi-Ref CD63-mNG EVs were acquired to evaluate dual fluorescence expression (mNG and CD63-AF647). Results Unstained Vesi-Ref CD63-mNG EVs showed optimal acquisition at 1:1,000 dilution, yielding approximately 2,164 events per second (EPS) and distinct separation from instrument noise. The mNG fluorescent population was detected in the B531-H channel and exhibited two discernible size populations based on VSSC1-H gating. Antibody-only controls for CD63-AF647 indicated minimal aggregation across all tested concentrations, with lower concentrations (0.25 µg/mL and 0.5 µg/mL) showing reduced background in the R670-H channel. Staining of Vesi-Ref CD63-mNG EVs with CD63-AF647 consistently resulted in approximately 72% dual-positive events (mNG and CD63-AF647) across all antibody concentrations, alongside a minor (3—4%) population positive for CD63-AF647 only (0.4%). Conclusion These findings confirm the suitability of the Vesi-Ref CD63-mNG EV reference standard as a positive control for EV characterization in nanoscale flow cytometry, supporting the validation of antibody-staining protocols due to its distinct fluorescent and scatter properties. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)