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One way of cells to communicate with each other is to release extracellular vesicles (EVs). EVs are phospholipid membrane enclosed nanoparticles that are released from cells. These tiny messengers are packed with a large range of molecules such as proteins, lipids and nucleic acids [1] (Hendricks et al. 2023). EVs can be very heterogenous depending on the context and cell type. These tiny vesicles contain valuable information that can reveal details about a cell’s condition. Since they are released to the surrounding environment, they can be captured and examined, which is particularly intriguing because the wealth of cellular information holds enormous promise as a diagnostic tool. The cell-to-cell communication via EVs can carry instructions and can therefore be used as prognostic markers, a way of delivering drugs and as therapeutic targets in various disease contexts.

The past years, research on EVs is steadily increasing and more than 5000 articles are published about EVs each year [2] (Théry Witwer et al. 2018). EV detection and characterization gives scientists the power to identify unique and personalized signatures in both healthy and sick patients, which potentially can answer questions scientists never even knew to ask in the first place.

Detecting EVs are a bit of a detective’s work. Scientists use special techniques, such as flow cytometry. Flow cytometry is an important technique to measure cells at a single cell level as well as their components such as surface molecules, nanoparticles or EVs. One of the many challenges with using flow cytometry for EV detection and characterization is their small size. In contrast to the normal range of detection of a regular flow cytometry (10-100μm), EVs belong to the nano-size range, mostly between 30-1000nm. However, advanced flow cytometers had led to easier immune-phenotyping and quantification of EVs, but careful validation and interpretation is needed, as EVs are small, heterogeneous and their concentrations can vary substantially.

To address this analysis gap, recent guides for EV detection using flow cytometry, suggest various ways to ensure rigor, standardization and reproducibility. Various Extracellular Vesicle Flow Cytometry Working groups recently published guidelines for improving EV detection by flow cytometry [3]. It includes recommendations for the minimal information to report when performing an experiment of EV detection using flow cytometry.

One of the important steps to ensure reproducibility and comparison between experiments is the adoption of reproducible and normalized protocols, allowing the comparison of data between experiments, devices and research groups worldwide. To achieve this, it is important to fully control the steps involved in EV-flow cytometry.

  1. Instrument calibration
  2. Experiment validation
  3. Sample preparation
  4. Sample measurement

The instrument calibration step includes all the manufacturer-defined steps to perform in order to reach good working conditions. This often involves a start-up procedure, fluidics clean-up and aligning optics for optimal performance [4]. Running particle standards is also frequently recommended as a final step to verify that the device is operating as expected.

Experiment validation is a process to make sure a device, such as flow cytometer, is capable of accurately performing a specific analysis. This is done by running a reference material that closely resembles the actual sample being tested, helping ensure the device’s reliability. In the context of EVs, Vesi-Ref CD63-GFP provides both the physical characteristics of an EV isolate (in size and number distribution) as well as the indication of the presence of key bio-chemical properties of extracellular vesicles: the presence of the tetraspanin protein CD63, via the expression of the fused-protein GFP, which is detectable by fluorescence. This step also includes running other sets of standards that normalize output (size in nm and concentration in particles/mL) as opposed to arbitrary units.

Once the EVs are isolated from for example urine or blood, it is important to characterize them in order to confirm their presence and identity.

Preparing the sample in such a way that it can be optimally detected in flow cytometry will dictate the quality of measurement regardless of the chosen flow cytometry platform. Whether the EV samples come from a cell culture isolate or from biofluids, it is important to obtain an indication of particle concentration before staining experiments. It is important to have an idea of the particle concentration, so that an optimal amount of reagent can be used for the labelling step, which typically includes a membrane dye, antibodies for protein phenotyping, and sometimes nucleic acid probes. Membrane dyes are typically very hard to solubilize, as they are very hydrophobic, and therefore tend to form micelles and aggregates in aqueous buffers. Recently, Vesi-Dye LMB-600 has been introduced as a water-soluble lipid membrane dye, alleviating such issues. Because of issues with steric hindrance, it is not advised to phenotype for the presence of too many protein surface markers at once. Depending on the experiment’s scope, it is possible to use a cocktail of anti-tetraspanin (CD9, CD63 & CD81) antibodies with similar fluorophores to estimate the concentration in “fully functional” EVs.

Another strategy is to simultaneously use an antibody against the most abundant of these 3 tetraspanin protein (membrane proteins in cells typically found on EVs) and one against the protein biomarker of interest.

The final strategy is to do away with phenotyping of CD9, CD63 or CD81 altogether, and to focus solely on specific exosomal markers for the given cell line or target organ, which could be on the surface or inside the EVs. This requires the use of antibodies of proven efficiency, primary labelled with a compatible fluorophore. Regardless of the dyes, probes and antibodies used, it is very important to remove any excess reagent before running the sample on a flow cytometer. Unlike cell analysis, which occurs far away from the instrument’s detection limit, EVs produce much weaker signals and securing an optimal signal-to noise ratio dramatically increases detection ability and reduces fluorescence background. So even a little bit of excess reagent could make your sample dirty and it is important to properly clean it, especially when working with EVs as their signals on a flow cytometer is very weak compared to cells. By running the sample post-incubation on a Vesi-SEC micro column, it is possible to remove excess reagent (small molecules, aptamers, nanobodies & antibodies) as well as residual proteins and aggregates. This is achieved quickly (1 minute centrifugation) and without significant dilution or product loss, unlike gravity size-exclusion chromatography. Thus prepared, samples can be analyzed in optimal conditions for flow cytometry. Running them at different dilution factors will ensure that swarm detection is not affecting the measurement. This point is achieved when event detection starts to decrease linearly with further dilution.

In summary, detecting EV and other nano bio particles involves a series of complex techniques to isolate, characterize and analyze them. By following the guidelines provided and using consistent methods and reagents it is possible to obtain reproducible data by using flow cytometry. If detected correctly, the information EVs carry can provide valuable insights into various diseases and may improve both diagnostics and therapeutics. An example of a recent advancement of EV research using flow cytometry found a way to distinguish brain cancer patients from healthy patients based on EVs isolated from the blood [5].

Frida Lind-Holm Mogensen

Neuro-Immunology Group, Department of Cancer Research

Luxembourg Institute of Health,
6A, rue Nicolas-Ernest Barblé,
L-1210 Luxembourg, Luxembourg
Faculty of Science, Technology and Medicine,
University of Luxembourg,
2, avenue de l’Université, L-4365
Esch-sur-Alzette, Luxembourg

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