Scientific Writer Beckman Coulter, Inc., Karnataka, India
Disclosure(s):
Anisha Jose, PhD: No financial relationships to disclose
Introduction/Rationale: 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.
