Precise analysis of single small extracellular vesicles using flow cytometry

Extracellular vesicles (EVs) are tiny bubble-like structures released by cells into the extracellular environment, playing crucial roles in intercellular communication and disease pathogenesis. Among these EVs, small extracellular vesicles (sEVs) are of particular interest due to their potential as diagnostic biomarkers and therapeutic targets. However, accurately quantifying and characterizing sEVs using flow cytometry, a powerful analytical tool, remains challenging.

A recent study by researchers at Kanazawa University delves into this challenge, evaluating four different preparation methods for flow cytometry analysis of sEVs. These methods include ultracentrifugation, density gradient centrifugation, size exclusion chromatography (SEC), and the TIM4-affinity method. The aim is to identify the most effective approach for specific and sensitive quantification of sEVs while minimizing non-specific binding and false-positive results.

One of the key issues in sEV flow cytometry analysis is the potential for non-specific binding of antibodies, leading to inaccurate quantification. The study compares the performance of control IgG and target-specific IgG antibodies and finds that they exhibit different binding characteristics, making direct comparison inappropriate.

AFM observation of sEVs washed by different methods

Figure 2

UC sEVs from 293T WT cells were stained with FITC-anti human CD9 antibody for 2 h. The stained sEVs were washed via ultracentrifugation (A: unstained, D: stained), SEC using qEV35 (B: unstained, E: stained), or TIM4-affinity method (C: unstained, F: stained). The sEVs were observed under an AFM, the scale bar represents 400 nm; a color tone shows height. A cross-section of a typical sEV and a typical background area with no sEV, indicated by a solid line and a dashed line respectively, is shown in the lower panel of AF.

The findings highlight that ultracentrifugation and density gradient centrifugation methods have high false-positive rates for tetraspanin staining, a common marker used for sEV identification. In contrast, SEC and the TIM4-affinity method show specific detection of single sEVs, providing more accurate and reliable results. These methods not only enable precise quantification of sEVs but also shed light on the roles of sEV biogenesis regulators in generating distinct sEV subpopulations.

Moreover, the study demonstrates the utility of SEC and the TIM4-affinity method for detecting rare disease-related markers, such as PD-L1, on sEVs. This opens up new possibilities for using sEV-based diagnostics and therapies in various diseases.

Overall, the development of reliable flow cytometry methods for sEV analysis represents a significant advancement in the field. These techniques have the potential to accelerate research into sEV biogenesis and facilitate the development of sEV-based diagnostic tools and therapeutic interventions for a wide range of diseases.

Kobayashi H, Shiba T, Yoshida T, Bolidong D, Kato K, Sato Y, Mochizuki M, Seto T, Kawashiri S, Hanayama R. (2024) Precise analysis of single small extracellular vesicles using flow cytometry. Sci Rep 14(1):7465. [article]

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