Nanovesicles can be found in the extracellular milieu, like tissue culture supernatant, but also in biological fluids, like plasma and urine, and they carry many types of biomolecules, including proteins, lipids, mRNA, miRNA and DNA 8. There are several databases including information on the content of EVs: Exocarta 4, EVPedia 5, Vesiclepedia 6, however, recent data have revealed that there is a great degree of heterogeneity among EVs and they exhibit different markers depending on the mechanism of release and the cellular origin 7. Other types of EVs include microvesicles, which are usually larger than exosomes (200 nm-1 µm) and do not originate from the endocytic pathway, instead they bud from the plasma membrane 3. There exist different types of EVs, among which the term exosomes refers to nanovesicles (30–200 nm) released after fusion with the plasma membrane of intraluminal vesicles enclosed in endocytic compartments known as multivesicular bodies (MVB) 1, 2. Most cell types release extracellular vesicles (EVs) during physiological processes. This method has the potential to allow any laboratory with access to conventional flow cytometry to identify surface markers on EVs, even non-abundant proteins, using minimally processed biological samples. However, whereas EVs isolated from 5–10 ml of urine were required for western blot detection of EpCAM, only 500 μl of urine were sufficient to visualise EpCAM expression by flow cytometry. In proof-of-concept experiments, an epithelial marker enriched in carcinoma cells, EpCAM, was identified in EVs from cell lines and directly in urine samples. We describe a straightforward method for EV detection using general EV markers like the tetraspanins CD9, CD63 and CD81, that allowed highly sensitive detection of urinary EVs without prior enrichment. To develop tools for EV analysis in biological samples, we evaluated here the critical parameters to optimise an assay based on immunocapture of EVs followed by flow cytometry. However, the lack of widespread, affordable methodologies for high-throughput EV analyses means that studies on biomarkers have not been done in large patient cohorts. EV biomarker detection requires highly sensitive techniques able to identify individual molecules. Extracellular vesicles (EVs) provide an invaluable tool to analyse physiological processes because they transport, in biological fluids, biomolecules secreted from diverse tissues of an individual.
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