Nano‐Flow Cytometry‐Guided Discrimination and Separation of Human Cytomegalovirus Virions and Extracellular Vesicles
Vladimir Bokun, Blair L. Strang, Paschalia Pantazi, Yan Liu, Beth Holder
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.