Characterization of Platelet and Red Blood Cell Extracellular Vesicles by Spectral Flow Cytometry.

We describe a method to characterize extracellular vesicles (EVs) derived from red blood cells (RBCs) and platelets. This chapter focuses on two widely used techniques: nanoparticle tracking analysis (NTA) and flow cytometry. NTA is particularly well suited to enumerating smaller particles (<200 nm), while flow cytometry techniques generally detect particles >200 nm. The advantage of flow cytometry detection of EVs is that the multiple fluorescent channels allow detection of markers on the surface EVs, which allows determination of their cell of origin. We present methods to isolate EVs for analysis or purify EVs for use in functional analyses based on differential centrifugation. Sensitivity for detection of EVs by flow cytometry varies widely from machine to machine, even within the same class of instruments. We demonstrate how to determine the sensitivity of small particle detection by flow cytometry and present representative data for quantification of RBC and platelet-derived EVs.