A Method for Isolation of Extracellular Vesicles and Characterization of Exosomes from Brain Extracellular Space
A Method for Isolation of Extracellular Vesicles and Characterization of Exosomes from Brain Extracellular Space
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DOI：
10.1007/978-1-4939-6728-5_10
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发表时间：
2017-01-01
期刊：
影响因子：
--
通讯作者：
Levy, Efrat
中科院分区：
文献类型：
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作者：
Perez-Gonzalez, Rocio;Gauthier, Sebastien A.;Levy, Efrat
细胞外囊泡（EV），包括外泌体、内吞来源的分泌囊泡和源自质膜的微泡，已从条件培养基和体液中广泛分离和表征。然而，从组织中分离 EV 的困难阻碍了他们的体内研究。在这里，我们描述了一种新方法，旨在从脑组织细胞外空间分离 EV 并表征外泌体。 EV 的纯化是通过温和地解离组织以释放大脑细胞外空间，然后依次进行低速离心、过滤和超速离心来实现的。为了进一步纯化 EV 与其他细胞外成分，在蔗糖梯度梯度上将它们分离。通过电子显微镜对蔗糖阶梯梯度组分的表征表明，该方法产生不含大囊泡、亚细胞细胞器或碎片的纯 EV 制剂。通过乙酰胆碱酯酶活性测定和总蛋白估计来确定 EV 分泌水平和含量，并通过蛋白质印迹和免疫电子显微镜分析外泌体鉴定和蛋白质含量。此外，我们在此提出了一种对 EV 进行脱脂的方法，以提高 EV 蛋白下游电泳分析的分辨率。
Extracellular vesicles (EV), including exosomes, secreted vesicles of endocytic origin, and microvesicles derived from the plasma membrane, have been widely isolated and characterized from conditioned culture media and bodily fluids. The difficulty in isolating EV from tissues, however, has hindered their study in vivo. Here, we describe a novel method designed to isolate EV and characterize exosomes from the extracellular space of brain tissues. The purification of EV is achieved by gentle dissociation of the tissue to free the brain extracellular space, followed by sequential low-speed centrifugations, filtration, and ultracentrifugations. To further purify EV from other extracellular components, they are separated on a sucrose step gradient. Characterization of the sucrose step gradient fractions by electron microscopy demonstrates that this method yields pure EV preparations free of large vesicles, subcellular organelles, or debris. The level of EV secretion and content are determined by assays for acetylcholinesterase activity and total protein estimation, and exosomal identification and protein content are analyzed by Western blot and immuno-electron microscopy. Additionally, we present here a method to delipidate EV in order to improve the resolution of downstream electrophoretic analysis of EV proteins.