Abstract

A Multi-Dimensional Approach to EV Flow Cytometry

Andries Zijlstra, Associate Professor of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center and Vanderbilt Ingram Cancer Center

Flow cytometry has proven to be very promising method for Single-EV analysis. Unfortunately, the heterogeneity of EV populations prevents the application of the single-particle classification strategy derived from conventional flow cytometry of intact cells fails in our attempts to resolve distinct classes of EVs. In simple terms, the small size of most EVs prevents the incorporation of all protein markers that define a uniform class of EVs. Consequently, EVs generated through the same biogenesis pathway from the same donor cells, could contain vastly different cargo. Conversely, restricting EV classification only to a size range is equally limiting. To improve EV sub-classification and enable the identification of biologically-significant alterations in EV populations in response to alterations in physiological or pathological states of the donor cell/tissue, we have developed a multi-dimensional approach to EV flow cytometry that attempts to cluster EVs into subpopulations on the basis of many, rather than single parameters.