Isolating Brain Mitochondria by Differential Centrifugation
差速离心法分离脑线粒体
发布: 2016年05月20日第6卷第10期 DOI: 10.21769/BioProtoc.1810 浏览次数: 17266
评审: Oneil G. BhalalaTifany DesprezHong Lok Lung
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参见作者原研究论文
The authors used this protocol in:
Feb 2015
Abstract
In addition to methods aimed at the study of mitochondrial function in-situ, a full understanding of mitochondrial function requires their purification from cells or tissues under specific physiological or pathological conditions. This protocol illustrates a sequential procedure to obtain functional mitochondria with high yield from mice brain tissue. Mitochondria obtained with this method can be used to assess different mitochondrial parameters, including oxygen consumption, membrane potential and calcium retention capacity.
Keywords: Mitochondria (线粒体)
Brain (脑)
Respiration (呼吸)
Bioenergetics (生物能学)
Mice (老鼠)
Materials and Reagents
- Centrifuge tubes
- Mice
- Sucrose (Sigma-Aldrich, catalog number: 84100 )
- Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A6003 )
- Ethylene glycol-bis(2-aminoethylether)-N, N, N’, N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E4378 )
- HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630-080 )
- Protease inhibitors (100x) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 78429 )
- Digitonin (Sigma-Aldrich, catalog number: D141 )
- D-Mannitol (Sigma-Aldrich, catalog number: M4125 )
- Magnesium chloride hexahydrate (MgCl2) (Sigma-Aldrich, catalog number: M9272 )
- Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 221473 )
- Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S5881 )
- Extraction buffer (approximately 50 ml per brain) (see Recipes)
Equipment
- Dounce homogenizer and pestles (A and B)
- Scissors
- Tweezers
Figure 1. Tools for mincing and homogenate the tissue. Dounce homogenizer and pestles (A and B), Small scissors and tweezers.
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Amigo, I., Traba, J. and Rueda, C. B. (2016). Isolating Brain Mitochondria by Differential Centrifugation. Bio-protocol 6(10): e1810. DOI: 10.21769/BioProtoc.1810.
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Rueda, C. B., Traba, J., Amigo, I., Llorente-Folch, I., Gonzalez-Sanchez, P., Pardo, B., Esteban, J. A., del Arco, A. and Satrustegui, J. (2015). Mitochondrial ATP-Mg/Pi carrier SCaMC-3/Slc25a23 counteracts PARP-1-dependent fall in mitochondrial ATP caused by excitotoxic insults in neurons. J Neurosci 35(8): 3566-3581.
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分类
神经科学 > 细胞机理 > 线粒体
细胞生物学 > 细胞器分离 > 线粒体
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