Shining a light on fluorescent EV dyes: Evaluating efficacy, specificity and suitability by nano‐flow cytometry

Extracellular vesicles (EVs) are mediators of intercellular communication, recently recognised for their clinical applications. Accurate characterisation and quantification of EVs are critical for understanding of their function and clinical relevance. Many platforms utilise fluorescence for EV char...

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Bibliographic Details
Published in:Journal of extracellular biology 2024-10, Vol.3 (10), p.e70006-n/a
Main Authors: Brealey, Joseph, Lees, Rebecca, Tempest, Robert, Law, Alice, Guarnerio, Sonia, Maani, Rawan, Puvanenthiran, Soozana, Peake, Nick, Pink, Ryan, Peacock, Ben
Format: Article
Language:English
Subjects:
brightness
extracellular vesicles (EVs)
fluorescent labelling
membrane dyes
nano‐flow cytometry
single particle characterisation
specificity
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Summary:Extracellular vesicles (EVs) are mediators of intercellular communication, recently recognised for their clinical applications. Accurate characterisation and quantification of EVs are critical for understanding of their function and clinical relevance. Many platforms utilise fluorescence for EV characterisation, frequently labelling surface proteins to identify EVs. The heterogeneity of EVs and the lack of a universal protein marker encourages the use of generic EV labelling methods, including membrane labelling. Using nano‐flow cytometry, we evaluated six membrane dyes, including MemGlow and CellMask. Evaluation criteria included EV labelling efficacy, non‐specific labelling of very low‐density lipoproteins (VLDLs), brightness and dye aggregation. Significant variation was observed in dye performance, with certain dyes showing poor EV labelling efficacy or high affinity to VLDLs. Importantly, several promising candidates were identified for further investigation. Overall, this study highlights the importance of selecting appropriate membrane dyes for EV staining tailored to the aims of the study and the EV origin. MemGlow and CellMask proved favourable, allowing bright, sensitive staining of EV membranes with minimal aggregation. However, MemGlow showed an affinity to VLDLs, and CellMask requires additional sample handling for optimal labelling. These results contribute to deepening our understanding of EV membrane dyes, allowing for better dye selection and EV identification in future studies.
ISSN:2768-2811
2768-2811
DOI:10.1002/jex2.70006