Flow Cytometry Protocols

John P. Nolan, Erika Duggan, Nolan, John P., Duggan, Erika

Extracellular vesicles (EVs) are released by cells and can be found in cell culture supernatants and biofluids. EVs carry proteins, nucleic acids, and other cellular components and can deliver these to nearby or distant cells, making EVs of interest as both disease biomarkers and therapeutic targets. EVs in biofluids are heterogeneous, coming from different cell types and from different sources with the cell, which limits the usefulness of bulk EV analysis methods that report the average features of all EVs present. Single-particle measurements such as flow cytometry would be preferred, but the small size and low abundance of surface antigens challenges conventional flow cytometry approaches, leading to the development of vesicle-specific assays and experimental design. Among the key issues that have emerged are: (a) judicious choice of detection (triggering) approach; (b) appropriate control experiments to confirm the vesicular nature of the detected events and the contribution of coincidence (aka swarm detection); and (c) the importance of fluorescence calibration to allow data to be compared over time and between laboratories. We illustrate these issues in the context of fluorescence-triggered Vesicle Flow Cytometry (VFC), a general approach to the quantitative measurement of EV number, size, and surface marker expression.