Shedding light on extracellular vesicles – a guide to labelling strategies

Extracellular vesicles (EVs) are tiny packages released by cells that carry various molecules, such as proteins and nucleic acids, playing critical roles in intercellular communication and disease progression. Studying these elusive entities requires sophisticated labelling techniques to track them accurately both in vitro and in vivo. In a recent article, researchers at Masaryk University have compiled a comprehensive overview of different labelling strategies for EVs, shedding light on their applications, strengths, and limitations.

The researchers cover a wide range of labelling methods, including fluorescent and bioluminescent labelling, which allow researchers to visualize EVs under a microscope. These techniques provide valuable insights into the behavior and interactions of EVs within biological systems. Additionally, the article discusses the use of radioactive tracers and contrast agents, enabling researchers to track EVs spatially and temporally in living organisms.

One of the highlights of the article is the exploration of endogenous labelling mechanisms, such as the Cre-lox and CRISPR-Cas systems. These cutting-edge technologies offer researchers the ability to label EVs directly within cells, providing real-time monitoring and tracking of EVs’ fate in target cells. Such approaches offer unprecedented opportunities for understanding the dynamics of EVs in complex biological environments.

By summarizing the latest advancements in EV labelling techniques, the review serves as a valuable resource for researchers seeking to select the most appropriate method for their EV-based studies. Whether investigating the role of EVs in disease pathogenesis or exploring their potential as therapeutic agents, choosing the right labelling strategy is crucial for accurate and insightful research outcomes.

This review underscores the importance of innovative labelling strategies in advancing our understanding of EV biology. By harnessing the power of various labelling techniques, researchers can unlock the secrets of EVs and harness their potential for diagnostic and therapeutic purposes. With continued advancements in labelling technologies, the future holds great promise for unraveling the mysteries of these tiny yet mighty vesicles.

Strategies for the labelling of endogenously produced EVs
for the study of their functional transfer

Fig. 3

A Cre-LoxP method. B CRISPR-Cas9 method. C Principle of Cre-LoxP system: Cre-mediated recombination activates a fluorescence switch from DsRed to eGFP in reporter+ cells after receiving Cre+-EVs produced by Cre+ cells. D Mechanism of CRISPR-Cas9: Reporter+ cells expressing Cas9 switch from mCherry to eGFP fluorescence after functional uptake of EVs. EVs produced by genetically manipulated donor cells carry a specific targeting single guide RNA (sgRNA) that navigates Cas9 with nuclease activity to generate cleavage of the target sequence

Boudna M, Campos AD, Vychytilova-Faltejskova P, Machackova T, Slaby O, Souckova K. (2024) Strategies for labelling of exogenous and endogenous extracellular vesicles and their application for in vitro and in vivo functional studies. Cell Commun Signal 22(1):171. [article]

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