Isolation of functional mitochondria from rat kidney and skeletal muscle without manual homogenization.
Isolation of functional mitochondria from rat kidney and skeletal muscle without manual homogenization.
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DOI：
10.1016/j.ab.2011.07.017
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发表时间：
2011-11-15
影响因子：
2.9
通讯作者：
Kristal BS
中科院分区：
文献类型：
--
作者：
Gross VS;Greenberg HK;Baranov SV;Carlson GM;Stavrovskaya IG;Lazarev AV;Kristal BS
从实体组织中分离功能完整的线粒体对于专注于阐明正常线粒体生理学和/或衰老、糖尿病和癌症等病症中的线粒体功能障碍的研究至关重要。人们越来越认识到线粒体作为药物开发的靶点和药物副作用的脱靶介质的重要性。不幸的是，从组织中分离线粒体通常使用基于均质器的方法进行，该方法需要广泛的操作者经验以获得可再现的高质量制备物。这些方法限制了传播，阻碍了扩大规模，并导致难以随着时间的推移和在不同实验室之间复制实验结果。在这里，我们描述了半自动化的方法来破坏组织，使用肾脏和肌肉线粒体制剂作为范例。这些方法使用Barocycler或PCT Shredder或两者。PCT Shredder是一种机械研磨机，可快速破碎组织，而不会产生过度均质化的重大风险。使用PCT Shredder分离的线粒体显示与对照相当。Barocycler产生受控的压力脉冲，可以调节以裂解细胞并释放细胞器。经受压力循环介导的组织破坏的线粒体显示出保留功能性，使得PCT Shredder和Barocycler的组合能够用于纯化线粒体制备物。
Isolation of functional and intact mitochondria from solid tissue is crucial for studies that focus on the elucidation of normal mitochondrial physiology and/or mitochondrial dysfunction in conditions such as aging, diabetes and cancer. There is growing recognition of the importance of mitochondria as both targets for drug development and as off-target mediators of drug side effects. Unfortunately, mitochondrial isolation from tissue is generally carried out using homogenizer-based methods that require extensive operator experience to obtain reproducible, high-quality preparations. These methods limit dissemination, impede scale-up, and contribute to difficulties in reproducing experimental results over time and across laboratories. Here we describe semi-automated methods to disrupt tissue, using kidney and muscle mitochondria preparations as exemplars. These methods utilize either a Barocycler, or The PCT Shredder, or both. The PCT Shredder is a mechanical grinder that quickly breaks up tissue without significant risk of over-homogenization. Mitochondria isolated using The PCT Shredder are shown to be comparable to controls. The Barocycler generates controlled pressure pulses that can be adjusted to lyse cells and release organelles. The mitochondria subjected to pressure cycling-mediated tissue disruption are shown to retain functionality, enabling combinations of The PCT Shredder and Barocycler to be used to purify mitochondrial preparations.
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影响因子：
3
作者：
Kristián, T;Hopkins, IB;Fiskum, G
通讯作者：
Fiskum, G
影响因子：
7.4
作者：
Stavrovskaya, Irina G.;Baranov, Sergei V.;Kristal, Bruce S.
通讯作者：
Kristal, Bruce S.
影响因子：
14.8
作者：
Frezza, Christian;Cipolat, Sara;Scorrano, Luca
通讯作者：
Scorrano, Luca
影响因子：
4.3
作者：
Skalska, Jolanta;Piwonska, Marta;Kunz, Wolfram S.
通讯作者：
Kunz, Wolfram S.
影响因子：
3.3
作者：
La-Salete, R.;Oliveira, M. M.;Peixoto, F. P.
通讯作者：
Peixoto, F. P.