Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons
Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons
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DOI：
10.3791/2704
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发表时间：
2011-05-01
影响因子：
1.2
通讯作者：
Bakowska, Joanna C.
中科院分区：
文献类型：
--
作者：
Joshi, Dinesh C.;Bakowska, Joanna C.
线粒体膜电位（Δ psi m）对于维持呼吸链产生ATP的生理功能至关重要。一个重大的损失.. M使细胞能量耗尽，随后死亡。活性氧（ROS）是重要的信号分子，但它们在病理条件下的积累导致氧化应激。细胞中ROS的两个主要来源是环境毒素和氧化磷酸化过程。线粒体功能障碍和氧化应激已被牵连在许多疾病的病理生理学;因此，确定的能力。m和ROS可以提供有关细胞生理状态和线粒体功能的重要线索。几种荧光探针（罗丹明123，TMRM，TMRE，JC- 1）可用于测定。m在多种细胞类型中，许多荧光指示剂（二氢乙锭、二氢罗丹明123、H2 DCF-DA）可用于测定ROS。几乎所有可用的荧光探针都用于评估.. m或ROS是单波长指示剂，其增加或减少其荧光强度与增加或减少的水平的刺激成比例。m或ROS。因此，必须在基线水平和施加特定刺激后测量这些探针的荧光强度。这允许人们确定基线水平和刺激之间的荧光强度变化的百分比。荧光强度的这种变化反映了相对水平的变化。m或ROS。在本视频中，我们演示了如何在大鼠皮层神经元中应用荧光指示剂TMRM，以确定TMRM荧光强度在基线水平和应用FCCP（线粒体解偶联剂）后的百分比变化。FCCP处理导致的较低水平的TMRM荧光反映了线粒体膜电位的去极化。我们还展示了如何应用荧光探针H2 DCF-DA来评估皮质神经元中的ROS水平，首先在基线处，然后在应用H2 O2之后。本方案（经微小修改）也可用于确定... m和ROS在不同类型的细胞和从其他脑区分离的神经元中的表达。
Mitochondrial membrane potential (Delta psi m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of..m renders cells depleted of energy with subsequent death. Reactive oxygen species ( ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine..m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. Several fluorescent probes ( Rhodamine 123, TMRM, TMRE, JC- 1) can be used to determine..m in a variety of cell types, and many fluorescence indicators ( Dihydroethidium, Dihydrorhodamine 123, H2DCF- DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess..m or ROS are single- wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of..m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of..m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF- DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol ( with minor modifications) can be also used to determine changes in..m and ROS in different cell types and in neurons isolated from other brain regions.