Quantification and characterization of extracellular vesicles by flow cytometry

Extracellular vesicles (EVs) are nano-sized, membrane-surrounded vesicles released by cells under both physiological and pathological conditions. Due to their small size and heterogeneity, comprehensive characterization of EVs remains technically challenging. Among the various analytical tools developed, flow cytometry stands out as a highly versatile and scalable platform, offering high-throughput analysis, multiparametric phenotyping, and quantitative detection. However, conventional flow cytometers are typically designed for cell-sized particles (0.5-40 µm) and require specific optimizations to reliably detect and analyze EVs, which are significantly smaller and result in weaker signals. These optimizations include instrument settings, sample handling and labelling strategies as well as acquisition protocols. Robust calibration and the use of appropriate controls are essential to ensure data accuracy and reproducibility across platforms. In this chapter, we outline the principles, technical considerations, and advantages of applying flow cytometry and imaging flow cytometry to EV research. We also highlight representative applications in both scientific and clinical contexts and discuss future directions for the field.