Extracellular vesicles (EVs): you know they are important, so make sure you can detect them!
The fields of virology, microbiology, nanoparticles, and extracellular vesicles have grown tremendously over the past few years.Extracellular vesicles are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies and are released under normal physiological conditions, as well as in the pathogenesis of neurological, vascular, hematological, and autoimmune diseases. Being able to detect and analyse such particles is therefore important. This article explores the effective options available from Luminex.
Extracellular vesicle, Imaging, Flow Cytometry, Small particles, Amnis
All cells, healthy or otherwise, release EVs as key mediators of intercellular communication. EVs are membrane derived structures that include exosomes, microvesicles, and apoptotic bodies. In particular, exosomes have been shown to transfer molecules between cells and potentially to transfer signals between cells. Exosomes are released under normal physiological conditions; however, they are also believed to serve as mediators in the pathogenesis of neurological, vascular, hematological, and autoimmune diseases, as well as cancer.
Therefore, it is not surprising that the analysis of such vesicles could provide insights into the mechanisms involved in normal activities and disease states. However, how can we do this with confidence? It is well known preparing and validating EVs is a technical challenge, not least of which because they are so small, but also because the classical means to detect and validate single vesicles rely on the use of fluorophores linked to antibodies. Although we may have specific antibodies available, the fact that EVs have far fewer surface markers means that the intensity of any fluorescence signal is often so low that it gets lost in the background.
Conventional approaches to understanding the role of EVs in disease and health, including microscopy, are often slow, whereas PMT-based flow cytometry, fall short on several fronts. Designed for particles larger than approximately 300 nm, they miss too many details—particularly smaller EVs due to their refractive indices or EVs with rare surface markers. If you are able to see the vesicles or gate away from the background noise then this must be a desirable capability.
Our Amnis flow cytometers including the Amnis CellStream Cytometer and the ImageStream X Mk II Imaging Flow Cytometer offer avenues to overcome such issues. Employing a charge-coupled device (CCD) using Time Delay Integration (TDI), offers the advantages of high-throughput flow cytometry as well high-sensitivity detection of submicron particles.
To identify potential EVs, a gate was set using an SSC vs. FSC plot (Figure 1A).
