Introduction: Extracellular vesicles (EVs) are key mediators of intercellular communication, enclosed by a phospholipid bilayer and lack the ability to replicate. Characterization of EVs is essential to elucidate their cellular origins, molecular cargo, and functional roles in health and disease. However, the nanoscale size and low refractive index (RI) present significant challenges for detection and size determination with conventional flow cytometers. Furthermore, the use of standard polystyrene beads for calibration complicates the analysis due to their significantly higher light-scattering properties compared to EVs. The nano flow cytometer represents a new platform for EV evaluation. The instrument is equipped with a multiple-scatter laser which increases the sensitivity for the detection of small EVs. By combining scatter-based detection with fluorescence antibodies labelling, size, concentration, and expression of EV surface markers can be measured simultaneously, providing a more accurate and sensitive approach to EV analysis.

Aim: The aim of this study was to calibrate the CytoFLEX nano flow cytometer for the detection and analysis of small EVs with diameters less than 300 nm. For this purpose, the Cellarcus Vesicle Analysis Kit (vFC™) and AcoDyes™ were employed. Following calibration, plasma-derived EVs were analyzed. Additionally, nanoViS polystyrene beads were used in combination with FCMPASS software to estimate the size distribution of EVs.

For Research Use Only. Not for use in diagnostic procedures.