Isolation and Characterization of Tissue and Cell-Derived Extracellular Vesicles and Non-Vesicular Extracellular Particles v2

Cells produce a heterogeneous complement of extracellular biomolecular complexes,including extracellular vesicles (EVs) and non-vesicular extracellular particles(NVEPs). EVs are lipid bilayer-enclosed membrane particles released by cells. NVEPsare recently discovered amembranous small multimolecular assemblies with distinctRNA and protein cargoes. EVs and NVEPs are released by most cell types and canbe found in biological fluids. Both types of extracellular complexes contain uniquecargo and have important biological functions. Due to their great potential asdiagnostic tools, there is a growing interest in being able to identify and characterizetissue- and cell- derived EVs and NVEPs. Here we described protocols for isolatingEVs by size exclusion chromatography (SEC) or an ultracentrifugation method tosimultaneously isolate EVs and NVEPs from conditioned media supernatant followingtissue or cell culture. In addition, we describe microfluidic resistive pulse sensing,vesicle flow cytometry, and immunoblotting methods for their characterization.