Nolan John P, Duggan Erika
Scintillon Institute, San Diego, CA, 92121, USA.
Methods Mol Biol. 2018;1678:79-92. doi: 10.1007/978-1-4939-7346-0_5.
Extracellular vesicles (EVs) are released by cells and can be found in cell culture supernatants and biofluids. EVs carry proteins, nucleic acids, and other cellular components and can deliver these to nearby or distant cells, making EVs of interest as both disease biomarkers and therapeutic targets. EVs in biofluids are heterogeneous, coming from different cell types and from different sources with the cell, which limits the usefulness of bulk EV analysis methods that report the average features of all EVs present. Single-particle measurements such as flow cytometry would be preferred, but the small size and low abundance of surface antigens challenges conventional flow cytometry approaches, leading to the development of vesicle-specific assays and experimental design. Among the key issues that have emerged are: (a) judicious choice of detection (triggering) approach; (b) appropriate control experiments to confirm the vesicular nature of the detected events and the contribution of coincidence (aka swarm detection); and (c) the importance of fluorescence calibration to allow data to be compared over time and between laboratories. We illustrate these issues in the context of fluorescence-triggered Vesicle Flow Cytometry (VFC), a general approach to the quantitative measurement of EV number, size, and surface marker expression.
细胞外囊泡（EVs）由细胞释放，可在细胞培养上清液和生物流体中找到。EVs携带蛋白质、核酸和其他细胞成分，并可将这些成分传递给附近或远处的细胞，这使得EVs作为疾病生物标志物和治疗靶点备受关注。生物流体中的EVs具有异质性，来自不同的细胞类型以及细胞内的不同来源，这限制了报告所有存在的EVs平均特征的整体EV分析方法的实用性。诸如流式细胞术之类的单颗粒测量方法会更受青睐，但表面抗原的小尺寸和低丰度对传统流式细胞术方法构成了挑战，从而促使了囊泡特异性检测方法和实验设计的发展。已出现的关键问题包括：（a）明智地选择检测（触发）方法；（b）进行适当的对照实验，以确认检测到的事件的囊泡性质以及巧合（即群体检测）的影响；（c）荧光校准的重要性，以便能够在不同时间以及不同实验室之间比较数据。我们在荧光触发的囊泡流式细胞术（VFC）的背景下阐述这些问题，VFC是一种用于定量测量EV数量、大小和表面标志物表达的通用方法。
