Factors to consider before choosing EV labeling method for fluorescence-based techniques.

A well-designed fluorescence-based analysis of extracellular vesicles (EV) can provide insights into the size, morphology, and biological function of EVs, which can be used in medical applications. Fluorescent nanoparticle tracking analysis with appropriate controls can provide reliable data for size and concentration measurements, while nanoscale flow cytometry is the most appropriate tool for characterizing molecular cargoes. Label selection is a crucial element in all fluorescence methods. The most comprehensive data can be obtained if several labeling approaches for a given marker are used, as they would provide complementary information about EV populations and interactions with the cells. In all EV-related experiments, the influence of lipoproteins and protein corona on the results should be considered. By reviewing and considering all the factors affecting EV labeling methods used in fluorescence-based techniques, we can assert that the data will provide as accurate as possible information about true EV biology and offer precise, clinically applicable information for future EV-based diagnostic or therapeutic applications.