Flow Cytometry for Single Extracellular Vesicle Analysis

Extracellular vesicles (EVs) are nanoscale lipid bilayer-enclosed vesicles secreted by cells to mediate intercellular communication via the transfer of proteins, nucleic acids, lipids, and metabolites. The particle size of EVs varies from approximately 30–1000 nm, with the majority falling below 200 nm. Because of the potential applications of EVs in disease diagnosis and therapy, their large inherent diversities in size, composition, and origin are highly appreciated. By providing multiparameter analysis of single particles in a high-throughput manner, flow cytometry (FCM) is essential for revealing the heterogeneity of EVs. However, the small size and small refractive index of EVs, limited quantity of cargo molecules, and lack of standardized EV measurements between instruments and laboratories have rendered such analyses challenging. In this chapter, we briefly introduce the principles and applications of conventional flow cytometers in the analysis of single EVs and discuss the difficulties faced when applying this technology. The development of ultrasensitive nano-FCM and its applications in single-EV analysis are also highlighted. Additionally, the importance of standardized reporting of EV FCM experiments is emphasized.