Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration
Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration
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DOI：
10.3791/51682
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发表时间：
2014-09-01
影响因子：
1.2
通讯作者：
McCully, James D.
中科院分区：
文献类型：
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作者：
Preble, Janine M.;Pacak, Christina A.;McCully, James D.
前面描述的线粒体分离方法使用差速离心法和/或Ficoll梯度离心法需要60到100分钟才能完成。我们描述了一种使用商用组织分离器和差异过滤从哺乳动物活检组织中快速分离线粒体的方法。在该方案中，组织分离器的标准化均质周期取代了人工均质。这允许组织的均匀一致的均质化，这是人工均质化不容易实现的。组织分离后，匀浆通过尼龙网状过滤器过滤，消除了重复的离心法步骤。因此，线粒体分离可以在不到30分钟的时间内完成。这种分离方法从0.18+/-0.04克(湿重)的组织样本中获得了大约2×10(10)活的、具有呼吸功能的线粒体。
Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator's standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 10(10) viable and respiration competent mitochondria from 0.18 +/- 0.04 g (wet weight) tissue sample.