Inglis Heather, Norris Philip, Danesh Ali
Blood Systems Research Institute.
Blood Systems Research Institute; Department of Medicine, University of California, San Francisco; Department of Laboratory Medicine, University of California, San Francisco;
J Vis Exp. 2015 Mar 17(97):52484. doi: 10.3791/52484.
Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal-to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.
细胞外囊泡（EVs）是存在于体液中的小的膜衍生囊泡，高度参与细胞间通讯，并有助于调节多种生物过程。由于其尺寸小且缺乏对感兴趣标记物呈阳性的离散群体，使用流式细胞术（FCM）分析EVs一直非常困难。尽管在过去十年中EV分析方法有了很大改进，但仍在不断完善。不幸的是，没有一种适用于所有情况的方案，在确定最合适的使用方法时必须考虑几个方面。本文介绍了几种处理EVs的不同技术以及两种使用单独检测或基于微珠方法分析EVs的方案。这里描述的方法将有助于消除商业制剂中常见的抗体聚集体，提高信噪比，并以合理的方式设置门控，以尽量减少背景荧光的检测。第一个方案使用单独检测方法，特别适合分析大量临床样本，而第二个方案使用基于微珠的方法来捕获和检测较小的EVs和外泌体。
