Fluorescent labeling of extracellular vesicles

Fluorescent labeling of extracellular vesicles (EVs) enables studying their uptake and influence on individual cells, biodistribution as well as facilitates their characterization using high-resolution flow cytometry at a single EV level. Here we describe the importance of fluorescent labeling, the available fluorescent dyes and labeling approaches, the characteristics of an ideal dye, and the available techniques for post-labeling purification. We discuss the importance of preserving the size of EVs for uptake, biodistribution, and characterization studies and focus on the effect of common lipophilic PKH and luminal CFSE dyes on the size of EVs. Lastly, we present an example protocol for luminal labeling of EVs and characterization of the effect of labeling on the size of EVs using nanoparticles tracking analysis (NTA).