Nano‐flow cytometry‐Guided discrimination and separation of human cytomegalovirus virions and extracellular vesicles
Author(s)
Bokun, Vladimir
Strang, Blair L
Pantazi, Paschalia
Liu, Yan
Holder, Beth
Type
Journal Article
Abstract
Accurate quantification and physical separation of viral particles and extracellular vesicles (EVs) produced by virus-infected cells presents a significant challenge due to their overlapping physical and biochemical properties. Most analytical methods provide information on a particle mixture as a whole, without distinguishing viral particles from EVs. By utilising nano-flow cytometry (nFC), a specialised form of flow cytometry adapted for the investigation of nanoparticles, we developed a simple, nucleic acid staining-based method for discrimination and simultaneous quantification of the human cytomegalovirus (HCMV) virions, dense bodies and EVs, within extracellular particle mixtures produced by HCMV-infected cells. We show that nucleic acid staining allows for discrimination of the individual particle types based on their distinct fluorescence/side scatter profiles, assessed at single-particle level by nFC. Following this, we optimised a method for physical separation of EVs from viral particles, based on high-speed centrifugation through density cushions, using nFC as a tool to evaluate the purity of the isolated EVs. The methods introduced here have the capacity to circumvent common difficulties associated with the co-investigation of EVs and viruses.
Date Issued
2025-05-01
Date Acceptance
2025-02-12
Citation
Journal of Extracellular Vesicles, 2025, 14 (5)
ISSN
2001-3078
Publisher
Wiley
Start Page
e70060
Journal / Book Title
Journal of Extracellular Vesicles
Volume
14
Issue
5
Copyright Statement
© 2025 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
License URL
Subjects
Humans
Cytomegalovirus
Virion
Cytomegalovirus Infections
Flow Cytometry
Extracellular Vesicles
Publication Status
Published
Article Number
e70060
Date Publish Online
2025-05-02