Techniques to improve detection and analysis of extracellular vesicles using flow cytometry.
Techniques to improve detection and analysis of extracellular vesicles using flow cytometry.
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DOI：
10.1002/cyto.a.22649
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发表时间：
2015-11
期刊：
影响因子：
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通讯作者：
Norris PJ
中科院分区：
文献类型：
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作者：
Inglis HC;Danesh A;Shah A;Lacroix J;Spinella PC;Norris PJ
细胞外囊泡（EVs）的大小从50 nm到1 μm不等。流式细胞术（FCM）是分析EVs最常用的方法；然而，由于电动汽车体积小且缺乏离散阳性人群，因此准确表征电动汽车仍然具有挑战性。在这里，我们报告了优化技术的使用，特别适合分析来自大量临床样品的ev。利用双管齐下的方法，包括1)预过滤抗体以去除聚集体，然后2)重复样品的洗涤剂裂解以解释剩余的假阳性事件，我们能够有效地限制假阳性非ev事件。此外，我们表明，裂解样品是一个有用的替代同种型设置门，以排除背景荧光。为了减少背景，我们开发了一种使用过滤器在染色后“清洗”样品的方法，从而提供了一种比超离心和蔗糖梯度分馏更快的替代方法。综上所述，这些优化技术的使用提高了FCM检测EV的准确性和效率。
Extracellular vesicles (EVs) range in size from 50 nm to 1 μm. Flow cytometry (FCM) is the most commonly used method for analyzing EVs; however, accurate characterization of EVs remains challenging due to their small size and lack of discrete positive populations. Here we report the use of optimization techniques that are especially well-suited for analyzing EVs from a high volume of clinical samples. Utilizing a two pronged approach that included 1) pre-filtration of antibodies to remove aggregates, followed by 2) detergent lysis of a replicate sample to account for remaining false positive events, we were able to effectively limit false positive non-EV events. In addition, we show that lysed samples are a useful alternative to isotypes for setting gates to exclude background fluorescence. To reduce background we developed an approach using filters to “wash” samples post-staining thus providing a faster alternative to ultracentrifugation and sucrose gradient fractionation. In conclusion, use of these optimized techniques enhances the accuracy and efficiency of EV detection using FCM.