Dedicated nanoparticle flow cytometry for single extracellular vesicle phenotyping: Performance of the CytoFLEX Nano

Accurate discrimination of extracellular vesicles (EVs) from non-vesicular nanoparticles and robust phenotyping of individual EVs directly within complex biofluids are essential to advance understanding of EV biology and to realize their potential as biomarkers and therapeutic agents. Conventional flow cytometers, originally designed for cellular analysis, lack the scatter and fluorescence sensitivity, dynamic range, and event-rate control required for quantitative characterization of nanoscale vesicles and are particularly susceptible to coincident (swarm) detection. Dedicated nanoparticle flow cytometers have been developed to address these limitations, and here we systematically evaluate the suitability of the CytoFLEX Nano (Beckman Coulter) for EV analysis. Using a series of calibration beads of different materials, fluorescent liposomes, and EVs isolated from peritoneal fluid and cell culture medium, we assess size-detection thresholds, scatter and fluorescence sensitivity, dynamic range, and volumetric counting accuracy. These data provide practical guidance for implementing nanoscale flow cytometry in EV research and support the informed adoption of dedicated nanoparticle cytometers in studies adhering to current EV reporting standards.