Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, MA 02118, USA.
Center for Network Systems Biology, Boston University School of Medicine, Boston, MA 02118, USA.
Methods. 2020 May 1;177:35-49. doi: 10.1016/j.ymeth.2020.02.002. Epub 2020 Feb 5.
Extracellular vesicle (EV) is a unified terminology of membrane-enclosed vesicular species ubiquitously secreted by almost every cell type and present in all body fluids. They carry a cargo of lipids, metabolites, nucleic acids and proteins for their clearance from cells as well as for cell-to-cell communications. The exact composition of EVs and their specific functions are not well understood due to the underdevelopment of the separation protocols, especially those from the central nervous system including animal and human brain tissues as well as cerebrospinal fluids, and the low yield of proteins in the separated EVs. To understand their exact molecular composition and their functional roles, development of the reliable protocols for EV separation is necessary. Here we report the methods for EV separation from human and mouse unfixed frozen brain tissues by a sucrose step gradient ultracentrifugation method, and from human cerebrospinal fluids by an affinity capture method. The separated EVs were assessed for morphological, biophysical and proteomic properties of separated EVs by nanoparticle tracking analysis, transmission electron microscopy, and labeled and label-free mass spectrometry for protein profiling with step-by-step protocols for each assessment.
细胞外囊泡（EV）是一种通用术语，指几乎所有细胞类型普遍分泌的、存在于所有体液中的膜封闭囊泡物种。它们携带脂质、代谢物、核酸和蛋白质等货物，用于细胞清除以及细胞间通讯。由于分离方案的欠发达，尤其是来自包括动物和人脑组织以及脑脊液的中枢神经系统的分离方案，以及分离的 EV 中蛋白质的产量低，因此 EV 的确切组成及其特定功能尚不清楚。为了了解其确切的分子组成及其功能作用，需要开发可靠的 EV 分离方案。在这里，我们报告了通过蔗糖分步超速离心法从人及鼠未固定的冷冻脑组织中分离 EV 的方法，以及通过亲和捕获法从人脑脊液中分离 EV 的方法。通过纳米颗粒跟踪分析、透射电子显微镜和标记和非标记质谱法，对分离的 EV 进行形态学、生物物理学和蛋白质组学特性评估，并提供了用于每个评估步骤的逐步方案。
