Analysis of individual extracellular vesicles by imaging flow cytometry

Abstract Virtually all cells release extracellular vesicles (EVs) into their environment, such as exosomes and microvesicles. EVs can mediate intercellular communication processes in a targeted manner. Representing their cell of origin, EVs contain cell type specific signatures, qualifying them as a novel class of biomarkers. Furthermore, according to their tropism to certain target cells, EVs provide promising aspects to be used as drug delivery vehicles. Depending on their origin, certain EVs contain the potential to modulate physiological and pathophysiological processes. Although the EV field provides many interesting aspects, the methodology in EV research is limited. For now, EVs are mainly analyzed by nanoparticle tracking analysis and bulk molecular analysis, regularly Western Blot. These technologies cannot dissect the heterogeneity of EVs observed by electron microscopy (EM). Although EM technologies help to demonstrate the heterogeneity within EV samples, EM technologies are not appropriate to perform more complex and quantitative EV analyses. Flow cytometry (FCM) is a traditional method for dissecting the heterogeneity of given cell populations in a quantitative and complex manner. However, classical FCM regularly fails to detect objects in the size range of small EVs (sEVs) that typically is in the range between 70 and 150 nm. Recently, we and others demonstrated the potential of imaging FCM for the analyses of small EVs at the single vesicle level. Here, at the example of sEVs harvested from supernatants of human mesenchymal stromal cells (MSCs), we share a protocol for studying the expression of the tetraspanins CD9, CD63 and CD81 on single EVs.