GO enrichment analysis was performed as described by refs

GO enrichment analysis was performed as described by refs. markers Fulvestrant S enantiomer for the analysis and isolation of different EV populations are missing, imposing important limitations to understanding Fulvestrant S enantiomer EV functions. Here, EVs from human dendritic cells were first separated by their sedimentation velocity, and then either by their behavior upon upward floatation into iodixanol gradients or by immuno-isolation. Considerable quantitative proteomic analysis allowing comparison of the isolated populations showed that several classically used exosome markers, like major histocompatibility complex, flotillin, and heat-shock 70-kDa proteins, are similarly present in all EVs. We recognized proteins specifically enriched in small EVs, and define a set of five protein categories displaying different relative large quantity in unique EV populations. We demonstrate the presence of exosomal and nonexosomal subpopulations within small EVs, and propose their differential separation by immuno-isolation using either CD63, CD81, or CD9. Our work thus provides guidelines to define subtypes of EVs for future functional studies. In the last decade communication between cells via secretion of membrane vesicles (collectively called extracellular vesicles or EVs) has become the focus of increasing interest (examined in refs. 1C3). EVs are composed of a lipid bilayer made up of transmembrane proteins and enclosing soluble proteins and RNA. The communication via EVs might be mediated by their multiple components. The surface receptors of EVs allow their targeting and capture by recipient cells, which can then incorporate proteic, lipidic, and even genetic messages carried by the vesicles, resulting in modifications of their physiological state (1, 4, 5). Consequently, the possibility of using these vesicles as biomarkers in various diseases, as vehicles of immunotherapies, or as targets to be eliminated to improve patients health is currently being explored (2, 6). It has become clear, however, in the past few years, that cells can release EVs of different types. Some EVs are directly created and released from your cells plasma membrane (PM), and are often called microparticles, (shed) microvesicles, or ectosomes: these EVs display sizes ranging from Fulvestrant S enantiomer a few dozens of nanometers to a few micrometers. Internal vesicles generated within multivesicular endosomal compartments (MVB) are secreted when these compartments fuse with the PM, thus releasing their internal vesicles in the extracellular milieu: these EVs are termed exosomes (3, 7). Exosomes CAMK2 are classically defined by their size, similar to the size of intraluminal vesicles of MVBs (i.e., below 150 nm in diameter), and their content of endosome-associated proteins. However, the term exosome is usually often used in the literature for EVs of small size, passing through 220-nm pore filters or recovered by high-speed ultracentrifugation (8), in the absence of demonstration of their intracellular origin. Such isolation procedures coisolate mixed EV populations, which we will call small EVs (sEVs), for lack of better term, in the rest of this article. Because EVs of different intracellular origins probably have different functional properties (9, 10), the mixed nature of EV preparations has made the growing literature increasingly confusing, with contradictory proposed functions and clinical uses of vesicles being regularly published. The lack of specific purification and characterization tools prevents a clear understanding of the specific versus shared functional properties of the different EVs. There is, therefore, a critical need for the identification of specific markers defining the different subtypes of EVs. To achieve this goal, we have performed here an extensive comparative analysis of the protein composition of all EVs recovered in the different steps of the differential ultracentrifugation (DUC) protocol classically used to isolate sEVs (11). As a source of EVs, we used human main monocyte-derived dendritic cells (DCs), whose exosomes and EVs have been described to promote immune responses (1, 12). We found that several proteins often used in the literature as exosome-markers, such as flotillin-1, heat-shock 70-kDa proteins.