Nano‐Flow Cytometry‐Guided Discrimination and Separation of Human Cytomegalovirus Virions and Extracellular Vesicles

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.