骨骼肌完整的线粒体的分离差速离心高分辨率测量呼吸运动
Summary
Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.
Abstract
Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.
Introduction
线粒体生物能量学和呼吸能力不仅可以在透化的细胞或纤维，而且在分离线粒体进行研究。在本研究中，我们描述了一个协议，使用隔离差速离心高分辨率呼吸测量测量完整骨骼肌线粒体。
为了分离完整的线粒体为呼吸测量，组织是均质和线粒体通过常规差速离心法分离。的差速离心的方法是基于连续的离心（在一系列增加速度的）组织匀浆首先由Pallade引入和同事近70年前1。将组织用剪刀第一切碎并在玻璃匀浆用宽松杵机械匀化。之后将匀浆在低速和包含完整的组织中得到的沉淀，细胞离心碎片和细胞核被丢弃。然后，将上清液在高速离心几次和线粒体富集馏分被收集。的差速离心方法的优点来分离线粒体是：i）该方法是快速的和线粒体可以1-1.5小时内（呼吸实验应尽可能快地进行）来分离; ii）其是便宜;和iii）它是非常有效的，并通过差速离心获得的线粒体是呼吸测量测定足够纯的。差速离心方法的缺点以分离线粒体是ⅰ）线粒体可能损坏和均化过程中脱开; ii）与其它细胞组分线粒体（的污染可能由进一步洗涤线粒体沉淀用另外的离心步骤）来解决; iii）选择不同的线粒体亚群， 例如 ，在差分离心步骤的可能性，麻省理工学院ochondria较低致密可以排除7;和iv）线粒体细胞周围缺少和仅理论最大呼吸可以被测量。分离线粒体为呼吸测量测定法的另一种方法是在密度梯度离心2。在该技术中，组织提取物层叠在蔗糖或Percoll密度梯度（具有较高密度在离心管的底部）中的溶液，并在一定的速度离心，使线粒体根据从其他细胞组分中分离出来的密度。这种方法经常被用于脑线粒体与来自突触体非常低的污染隔离。然而，通过密度梯度离心分离大鼠肝线粒体是高度污染的其他细胞器3。这种方法的局限性是，存在于离心管中的蔗糖梯度可能破裂，以便我线粒体（渗透冲击）。
根据组织的类型;还有要考虑完整的线粒体通过差速离心分离有些重要的因素。首先需要在一个温和的方式来组织均匀。软组织，如肾，脑和肝需要均化期间施加温和的机械力。与此相反，硬组织如需要更强的机械力心肌和骨骼肌。组织糜通常与蛋白酶的同质软化组织之前处理。均化和离心分离期间使用的所有缓冲液应该是冰冷的，并有一个生理相关pH与胞浆4,5兼容离子和渗透强度。
一项研究中分离线粒体生物能量学的一个优点是，蜂窝质膜不需要是permeabilized用洗涤剂如毛地黄皂苷或皂苷4,6，这可能会危及线粒体外膜的完整性。分离的线粒体的另一个优点是不存在的其他胞质因子，这可能与线粒体功能，如氧消耗干扰分析。使用分离的线粒体的缺点是某些线粒体种群在离心步骤的可能的选择，均化过程中损坏线粒体，并且为了获得分离的线粒体7,8的良好产率的高量生物样品的要求。
分离过程后，线粒体复合物的呼吸速率I-，II-和IV依赖性（状态2，3和4）使用高分辨率呼吸测量确定的。对于复杂的I-驱动的呼吸，谷氨酸和苹果酸中，随后加入二磷酸腺苷（ADP）。对于复杂的Ⅱ-驱动呼吸，琥珀酸盐，然后加入由ADP。为复合物IV驱动呼吸，抗坏血酸和tetramethylphenylendiamine（TMPD）接着加入ADP 9，10，11，12。状态2是指氧消耗在单独基板的存在。状态3是指氧消耗在衬底和ADP的存在。国家4指的耗氧量ADP耗尽后。的呼吸控制率（RCR）是氧消耗ATP产生的耦合的索引，并且被计算为状态3和状态4 13，15之间的比率。
总之，我们描述了一种协议通过差速离心分离功能和完整的骨骼肌线粒体和使用这些分离mitochondria用于功能和生物能量研究，例如高分辨率呼吸测量。
Protocol
Representative Results
Discussion
在本研究中，我们描述一个协议以分离高品质的，完整和差速离心紧密耦合骨骼肌线粒体可用于功能性研究，例如高分辨率呼吸测量。
为了分离完整和紧耦合线粒体，有要在本协议中考虑的一些关键点。收获骨骼组织后，应立即浸入冰冷线粒体隔离缓冲器。所有离心步骤应在4℃进行，并线粒体悬浮液应该总是在冰上分离过程中被保持。分离过程应尽可能快地完成。将匀?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This study was supported by the Swiss National Science Foundation (Grant 32003B_127619).
Materials
| ADP | Sigma | A 4386 | Chemical |
| Antimycin A | Sigma | A 8674 | Chemical, dissolve in ethanol |
| Ascorbate | Merck | 1.00127 | Chemical |
| ATP | Sigma | A 7699 | Chemical |
| BSA | Sigma | A 6003 | Chemical |
| EGTA | fluka | 3779 | Chemical |
| Glutamate | Sigma, | G 1626 | Chemical |
| Hepes | Sigma | H 7523 | Chemical |
| KCl | Merck | 1.04936 | Chemical |
| KH2PO4 | Merck | 1.04873 | Chemical |
| K-lactobionate | Sigma | L 2398 | Chemical |
| MgCl2 | Sigma | M 9272 | Chemical |
| Morpholinopropane sulphonic acid (MOPS) | Merck | 1.06129 | Chemical |
| O2k-Core: Oxygraph-2k | Oroboros Instruments | 10000-02 | High-resolution respirometry instrument |
| Proteinase, bacterial | Sigma | P 8038 | Chemical |
| Sodium azide | Sigma | S2002 | Chemical |
| Rotenone | Sigma | R 8875 | Chemical, dissolve in ethanol |
| Succinate | Sigma | S 2378 | Chemical |
| Schuett homogen-plus semiautomatic homogeniser | schuett-biotec GmbH | 3.201 011 | Tissue homogenizer |
| Taurine | Sigma | T 8691 | Chemical |
| TMPD | Sigma | T 3134 | Chemical |
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