Comparing Apples to Apples: Analysis of Extracellular Vesicles by Flow Cytometry

Extracellular vesicles (EVs) including exosomes and microvesicles are submicrometer-sized biological vesicles released by all cells, and can be found in all body fluids or harvested from cell culture supernatants. It is nowadays widely accepted that EVs can serve as vesicular messengers in various physiological and pathophysiological contexts. Over the last 10–15 years, the EV research community has grown exponentially and the field has attracted a lot of attention following numerous studies connecting EVs to therapeutic approaches such as vaccination, antitumor therapy, immunomodulation and drug delivery. Since the protein composition and cargo of EVs is assumed to resemble the cell releasing them, EVs also have come into focus as potential diagnostic biomarkers.

Due to their heterogenous nature and their small diameter (<200 nm), it is challenging to accurately measure individual EVs, and to quantify basic parameters such as diameter, size, and concentration of EVs in a sample of interest. In this context, flow cytometry is increasingly used to detect, quantify and phenotype EVs, and newer high-sensitivity flow cytometers have become available and are explored to measure EVs. Current challenges, however, comprise sensitivity, standardization (despite general lack of appropriate standards) and comparability of obtained data. I will give an introduction into possibilities and share examples and experiences in context of EV analysis by flow cytometry in various contexts.