Isolation of mitochondria-derived mitovesicles and subpopulations of microvesicles and exosomes from brain tissues.
Isolation of mitochondria-derived mitovesicles and subpopulations of microvesicles and exosomes from brain tissues.
复制标题
DOI：
10.1038/s41596-022-00719-1
复制
发表时间：
2022-11
期刊：
影响因子：
14.8
通讯作者：
Levy, Efrat
中科院分区：
文献类型：
--
作者：
D'Acunzo, Pasquale;Kim, Yohan;Ungania, Jonathan M.;Perez-Gonzalez, Rocio;Goulbourne, Chris N.;Levy, Efrat
细胞外囊泡（EVs）是由所有细胞类型分泌到细胞外空间的纳米级囊泡，包括大脑中的神经元和星形胶质细胞。电动汽车在生理和病理生理过程中发挥关键作用，如废物清除，细胞间通讯和运输保护性或致病性物质到细胞外空间。在这里，我们描述了一种详细的方案，用于从小鼠和人类大脑中可靠和一致地分离EV，适用于具有基本实验室经验的任何人，总时间为27小时。该方法包括脑组织的温和细胞外基质消化，一系列过滤和离心步骤来纯化EV，以及基于碘沙醇的高分辨率密度阶梯梯度，用于分离不同的EV群体，包括有丝分裂囊泡，一种新发现的线粒体起源的EV。我们还报告了详细的下游方案，用于表征和分析脑EV制剂，使用纳米轨道分析，电子显微镜和western blotting，以及测量有丝泡ATP动力学。此外，我们将这种新的基于碘二醇的高分辨率密度阶跃梯度与先前描述的基于蔗糖的梯度进行了比较。尽管回收的总EV产量相似，但与蔗糖梯度相比，基于碘二醇的梯度可以更好地分离不同的EV物种，包括微囊泡、外泌体和有丝分裂囊泡亚群。该技术允许在正常生理过程和病理脑条件下，包括神经退行性疾病，如阿尔茨海默病和帕金森病，对脑EV亚型进行定量、高度可重复的分析。该方案描述了从脑组织中分离细胞外囊泡（EV）亚群，包括微囊泡，外泌体和线粒体来源的有丝分裂囊泡，使用高分辨率（碘二醇）密度阶跃梯度。还介绍了EV的表征和分析。
Extracellular vesicles (EVs) are nanoscale vesicles secreted into the extracellular space by all cell types, including neurons and astrocytes in the brain. EVs play pivotal roles in physiological and pathophysiological processes such as waste removal, cell-to-cell communication and transport of either protective or pathogenic material into the extracellular space. Here, we describe a detailed protocol for the reliable and consistent isolation of EVs from both murine and human brains, intended for anyone with basic laboratory experience and performed in a total time of 27 h. The method includes a mild extracellular matrix digestion of the brain tissue, a series of filtration and centrifugation steps to purify EVs and an iodixanol-based high-resolution density step-gradient that fractionates different EV populations, including mitovesicles, a newly identified type of EV of mitochondrial origin. We also report detailed downstream protocols for the characterization and analysis of brain EV preparations using nanotrack analysis, electron microscopy and western blotting, as well as for measuring mitovesicular ATP kinetics. Furthermore, we compare this novel iodixanol-based high-resolution density step-gradient to the previously described sucrose-based gradient. Although the yield of total EVs recovered is similar, the iodixanol-based gradient better separates distinct EV species compared to the sucrose-based gradient, including subpopulations of microvesicles, exosomes and mitovesicles. This technique allows for quantitative, highly reproducible analyses of brain EV subtypes under normal physiological processes and pathological brain conditions, including neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. This protocol describes the isolation from brain tissue of extracellular vesicle (EV) subpopulations, including microvesicles, exosomes and mitochondria-derived mitovesicles, using a high-resolution (iodixanol) density step-gradient. EV characterization and analysis are also presented.
登录
查看更多内容
影响因子：
5.5
作者：
Kim KM;Meng Q;Perez de Acha O;Mustapic M;Cheng A;Eren E;Kundu G;Piao Y;Munk R;Wood WH 3rd;De S;Noh JH;Delannoy M;Cheng L;Abdelmohsen K;Kapogiannis D;Gorospe M
通讯作者：
Gorospe M
影响因子：
14.8
作者：
Brewer, Gregory J.;Torricelli, John R.
通讯作者：
Torricelli, John R.
影响因子：
2.3
作者：
Liu S;Hossinger A;Göbbels S;Vorberg IM
通讯作者：
Vorberg IM
影响因子：
16
作者：
Crescitelli, Rossella;Lasser, Cecilia;Lotvall, Jan
通讯作者：
Lotvall, Jan
影响因子：
3
作者：
D'Acunzo, Pasquale;Hargash, Tal;Levy, Efrat
通讯作者：
Levy, Efrat