![]() ![]() EVs range in size from small EVs (sEVs 200 nm), including microvesicles and large oncosomes, which are shed from the plasma membrane 3, 6, 7, 9, 11. However, it is becoming increasingly clear that different classes of EVs may contain specific cargo and, equally important, that NVEPs, such as exomeres and supermeres, contain many of the biomolecules, including proteins, RNA and DNA, that have previously been ascribed to exosomes 3, 9, 15, 16. Furthermore, the field has largely focused on studies related to EVs, while studies on extracellular amembranous NVEPs, including the recently discovered exomeres 5, 10 and supermeres 13, 14, 15, are limited. However, a major challenge in the field of EVs and NVEPs is their heterogeneity and the methods used to isolate and purify distinct populations 3, 5, 6, 7, 8, 9, 10, 11, 12. ![]() Overall, this protocol will allow those interested in isolating EVs and extracellular particles to advance scientific inquiry to answer outstanding questions in the field.Įxtracellular vesicles (EVs) and nonvesicular (NV) extracellular nanoparticles (NVEPs) play pivotal roles in both physiological and pathological conditions 1, 2, 3, 4. Adoption of this protocol will help translational investigators identify potential circulating biomarkers and therapeutic targets for a host of human diseases and allow basic scientists to better understand EV and NVEP biogenesis and function. The protocol can be applied to purification of EVs and NVEPs from cell culture medium and human plasma and requires ~72 h to complete. Compared to other isolation methods, our protocol has unique advantages, including high purity and reproducibility, with minimal expertise required. This protocol allows assignment and enrichment of a biomolecule of interest to its specific extracellular compartment. We describe in detail the use of differential ultracentrifugation, filtration, concentration and high-resolution density-gradient fractionation to obtain purified fractions of distinct populations of EVs and NVEPs. Here, we provide a comprehensive step-by-step protocol for sequential isolation of large and small EVs, nonvesicular fractions, exomeres and supermeres from the same starting material. At this juncture, it is critically important to have robust and reliable methods to separate distinct populations of EVs and NVEPs to assign cargo to their correct carrier. The EV field has largely focused on EV isolation and characterization, while studies on NVEPs are limited. In addition, two nonvesicular extracellular nanoparticles (NVEPs), exomeres and supermeres, have been discovered recently that are enriched in many cargo previously ascribed to EVs. There is an increasing appreciation for the heterogeneous nature of extracellular vesicles (EVs). ![]()
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