| Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement
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Author:
Date:
2016-08-05
[Abstract] The mitochondrial pathway of apoptosis involves a complex interplay between dozens of proteins and lipids, and is also dependent on the shape and size of mitochondria. The use of cellular models in past studies has not been ideal for investigating how the complex multi-factor interplay regulates the molecular mechanisms of mitochondrial outer membrane permeabilization (MOMP). Isolated systems have proven to be a paradigm to deconstruct MOMP into individual steps and to study the behavior of each subset of MOMP regulators. In particular, isolated mitochondria are key to in vitro studies of the BCL-2 family proteins, a complex family of pro-survival and pro-apoptotic proteins that directly control the mitochondrial pathway of apoptosis (Renault et al., 2013).
In ...
[摘要] 凋亡的线粒体途径涉及数十种蛋白质和脂质之间的复杂相互作用，并且还依赖于线粒体的形状和大小。在过去的研究中使用细胞模型不是理想的调查如何复杂的多因素相互作用调节线粒体外膜透化（MOMP）的分子机制。分离系统已被证明是将MOMP解构成各个步骤并研究每个子集的MOMP调节剂的行为的范例。特别地，分离的线粒体是BCL-2家族蛋白的体外研究的关键，BCL-2家族蛋白是直接控制凋亡的线粒体途径的促存活和促凋亡蛋白的复合家族（Renault > et al 。，2013）。
在这个协议，我们描述三个补充程序用于实时调查使用孤立的线粒体MOMP调节器的影响。第一种方法是"肝线粒体分离"，其中从小鼠中分离肝脏以获得线粒体。 "用JC-1和大小分级分离的线粒体标记"是描述标记，按大小分级并标准化线粒体亚群的方法的第二个方法。最后，"实时MOMP测量"协议允许在孤立的线粒体上实时跟踪MOMP。上述程序用于在体外确定线粒体膜形状在分离的细胞和分离的线粒体水平上的作用（Renault等人，2015）。
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| Arabidopsis thaliana Embryo Sac Mitochondrial Membrane Potential Stain
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Author:
Date:
2014-05-20
[Abstract] The aim of this experiment is to study mitochondrial functional status in Arabidopsis embryo sacs using the membrane potential indicator JC-1. Changes in the membrane potential are presumed to be due to the opening of the mitochondrial permeability transition pore (MPTP), allowing passage of ions and small molecules. The resulting equilibrium of ions leads in turn to the decoupling of the respiratory chain and the release of cytochrome c into the cytosol, a distinctive feature of the early stages of programmed cell death.
JC-1 is a lipophilic dye that can selectively enter into mitochondria and reversibly change color from green to red as the membrane potential increases. In healthy cells with high mitochondrial potential, JC-1 spontaneously forms complexes with ...
[摘要] 该实验的目的是使用膜电位指示剂JC-1研究拟南芥胚胎囊中的线粒体功能状态。假定膜电位的变化是由于线粒体通透性转换孔（MPTP）的打开，允许离子和小分子通过。所产生的离子平衡又导致呼吸链的解耦和细胞色素c释放到细胞溶质中，这是程序性细胞死亡的早期阶段的显着特征.JC-1是一种亲脂性染料，其可以选择性地进入线粒体并随着膜电位增加可逆地将颜色从绿色改变为红色。在具有高线粒体电位的健康细胞中，JC-1自发形成具有强烈红色荧光的复合物。另一方面，在具有低线粒体电位的线粒体中，JC-1保持单体形式，其仅显示绿色荧光（Martin等人，2013; Hauser等人， em，2006）。
此协议可用于孤立的线粒体，以及各种细胞类型和植物和其他生物体的不同组织。
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