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.
Keywords: 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).
Figure 1: Gating strategy for identifying EVs. A) Gating on potential EVs using FSC vs. SSC plot. B) Using the “Potential EVs” gated population, gate on PE+ events. The objects in the PE+ gate are the EVs labeled with CD235ab-PE.
C) Using the “Potential EVs” gated population, gate on the APC+ events. The objects in the APC+ gate are EVs labeled with CD41-APC.
Using this “Potential EVs” gated population, (B) PE-positive (PE+), and (C) APC-positive (APC+) events were gated. Objects in the PE+ gate were the EVs labeled with CD235ab-PE, and objects in the APC+ gate were EVs labeled with CD41-APC.
The Amnis® TDI CCD camera technology preserves sensitivity and image quality, even with fast-moving particles, and captures multiple colors of fluorescence, as well as FSC and SSC with superior photonic sensitivity. The effect is similar to physically panning a camera. TDI avoids image streaking despite signal integration times that are orders of magnitude longer than those of conventional flow cytometry. So, if you want to take a look at your events, this technology provides the means to visually verify and is important to make sure that you are looking at real single events rather than debris or false signals.
Like any well-designed experiment, the use of appropriate controls is essential to the success of the study. Control samples include antibody and buffer only, detergent controls, EV control preparations as well as the use of serial dilutions to verify the detection of single EV particles and confirm swarm detection was not occurring, are essential. If single EV particles are being detected, the positive EV events will linearly decline while the fluorescence intensity of the positive events remains constant.
Incorrect particle phenotyping caused by coincident events, i.e., swarming, has the potential to lead to false conclusions about the biology of EVs. Quantitative features, utilizing the image collection capability of the ImageStream®X Mk II in IDEAS Software and using the CellStream can help to identify these false positive events.
The Amnis® range available through Luminex Corporation (a Diasorin Company), is a widely used and respected portfolio that has enabled many researchers to study EVs accurately and effectively. To find out more about the range and selected publications and application notes, please visit our website: https://www.luminexcorp.com/flow-cytometry-and-imaging/