Bokun Vladimir, Strang Blair L, Pantazi Paschalia, Liu Yan, Holder Beth
Institute of Reproductive and Developmental Biology, Department of Metabolism, Digestion and Reproduction, Faculty of Medicine, Imperial College London, London, UK.
Institute for Infection and Immunity, City St George's, University of London, London, UK.
J Extracell Vesicles. 2025 May;14(5):e70060. doi: 10.1002/jev2.70060.
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.
由于病毒颗粒和病毒感染细胞产生的细胞外囊泡（EVs）在物理和生化特性上存在重叠，因此对它们进行准确的定量分析和物理分离是一项重大挑战。大多数分析方法提供的是关于颗粒混合物整体的信息，无法区分病毒颗粒和细胞外囊泡。通过利用纳米流式细胞术（nFC），一种适用于研究纳米颗粒的特殊形式的流式细胞术，我们开发了一种基于核酸染色的简单方法，用于区分和同时定量人巨细胞病毒（HCMV）感染细胞产生的细胞外颗粒混合物中的HCMV病毒体、致密体和细胞外囊泡。我们表明，核酸染色能够基于单个颗粒的荧光/侧向散射特征区分不同类型的颗粒，通过纳米流式细胞术在单颗粒水平进行评估。在此基础上，我们优化了一种基于密度梯度高速离心从病毒颗粒中物理分离细胞外囊泡的方法，并使用纳米流式细胞术作为工具来评估分离得到的细胞外囊泡的纯度。本文介绍的方法能够克服与细胞外囊泡和病毒共同研究相关的常见困难。
