隔离和线粒体从培养细胞和小鼠组织功能分析
Summary
Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.
Abstract
Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.
Introduction
活细胞中的脂肪和碳水化合物的形式银行代谢能量，并使用该能量为生物合成，膜运输和移动。在胞质溶胶中通过饮食糖酵解在转换直接转化为ATP，获得一定的能量。然而，ATP产生的在细胞中的主要来源是在线粒体内通过线粒体呼吸链1利用。线粒体的体系结构提供了必要的空间取向为有效和高效的ATP的产生。线粒体具有一个双层膜由间空间，并与基体中，最里面的线粒体隔室，房屋一起的组分分离，并协调参与ATP生成的化学反应。内膜包含一系列膜结合蛋白复合物组成的呼吸链，以及ATP合酶中，蛋白质复合物带来的ADP和Pi一起用于形成ATP的。客栈ER膜被折叠成嵴和电子沿着经由细胞色素c，其中复合物之间移动间空间内的可溶电子载体呼吸链复合物通过。作为电子移动，还原当量发生氧化和氢离子从基质向间空间泵送。作为高离子浓度的间空间内的结果，电化学梯度建立导致跨线粒体内膜（Δψ）2的膜电位。氧气是电子传递链的最终电子受体，和氢离子从间空间流过的ATP合酶回矩阵，并在这样做的直接导致ATP的形成。这个过程的全部描述被称为氧化磷酸化。嵴的褶皱增加内层膜的表面积，从而允许内最大电子传输和ATP生产每个线粒体。的蛋白质，酶和参与氧化磷酸化的其它分子衍生自两个核和线粒体基因。线粒体含有自己的环状DNA，编码13种蛋白质，以及tRNA和必要的ATP生产3的mRNA。然而，许多更多的蛋白质是必需的，因此是核编码。大部分这些核编码蛋 白质定位到线粒体基质通过在所述前体蛋白的N-末端前序列使用的，它们的进口是由Δψ4,5-驱动部分。
超出促进细胞的生物能学，线粒体也影响主要的代谢过程，例如三氯乙酸和β-氧化，通过调节钙的蜂窝信令，以及在细胞凋亡6的关键作用。具体地说，在细胞应激时，驻留在或BCL-2家族蛋白在线粒体外膜相互作用可引起线粒体外膜渗透性（MOMP）7,8。期间的MOMP，细胞色素C和其他蛋白质被释放到细胞溶胶，并连同几个胞质蛋白形成复合物称为凋亡体9,10。的凋亡体激活胱天蛋白酶是继续在细胞凋亡的执行阶段裂解细胞蛋白质和DNA。一旦发生MOMP，ΔΨ被折叠和ATP的生产暂停。因此，如凋亡启动线粒体功能受到损害，并变化ΔΨ可以关联到线粒体和细胞的健康12。而凋亡是许多疾病模型，线粒体功能和改变ΔΨ端点也可以产生有关的疾病产生和/或进展的有价值的信息。例如，线粒体的结构和功能的变化已经神经变性疾病13,14的过程中记录。
在协议中，异的第一部分完整的线粒体是保留其ΔΨ的LATION描述。 HEK-293T细胞暴露于不同浓度的重组TNF-α，IL1-β和IFN-γ的组合来诱导细胞凋亡。选择这些细胞因子，因为它们经常报道为高的原代人败血性样品15和凋亡的外源性途径中可以通过TNF-α与其受体的结合6的相互作用触发。因为有必要以比培养细胞从主组织隔离官能线粒体微妙的变化，并且由于大量的研究利用动物，该协议还描述了如何从肝脏和前侧索硬化症（ALS）的小鼠模型的脊髓分离线粒体。
该协议的第二部分被开发利用的电位敏感的荧光染料用荧光板读数器监测扰动对线粒体膜电位。差异蜂窝状态（ 即 ，健康与不健康的）之间s的通过定量与解偶联剂，呼吸链抑制剂，复杂抑制剂和离子载体，所有这些都导致线粒体膜电位的耗散一起分离线粒体的ΔΨ的强度来区分。的线粒体健康，较大处理后在ΔΨ的变化与线粒体抑制剂，因此线粒体的反应可以用作线粒体（DYS）功能的一个指标。
采用分离线粒体，而不是在原地功能评估提供了确切的证据表明，病理或治疗直接调制改变细胞器16-18。虽然有在文献中以分离从培养的细胞中线粒体的方法，它们是模糊的17和/或利用专门的设备16。该协议详细描述了隔离的方法，是易于适应其它细胞系，包括初级和组织培养13,14,19,20。许多分离的线粒体的研究利用在这个协议，但与克拉克电极中使用的相同的线粒体解偶联剂和抑制剂（ 图21是多篇论文在文献中有代表性的例子），而这又是一个非常具体的和专门的设备。此外，这种传统的方法具有如低产量和高复杂22,23限制，并且需要大量的线粒体的（〜500微克/反应）。在这个协议中，该荧光膜感应电位探针TMRE结合使用用荧光板读数器，这是一个标准的机器在许多实验室。 TMRE被广泛认为是因为它快速进入细胞并分离线粒体，可以在低浓度24中。多个反应可以迅速地设置在串联和使用此协议批分析。此外，反应s需要一个更少量孤立的线粒体（〜10微克/反应）。通过要求较少的材料，更小的组织或细胞培养物的样品可以用作一个起点线粒体隔离，更多的重复或反应可以建立起来，并有可能足够的材料为其它分离线粒体实验如ATP产生，耗氧量，或导入分析是可能的。
Protocol
Representative Results
Discussion
HEK-293T细胞用重组细胞因子的治疗导致适量的细胞死亡超过48小时（ 图1）。诱导的TNF-α处理的细胞死亡的量是类似于以前报道的研究30和多种细胞因子是大于总结性的量与任何细胞因子的共施用后细胞活力降低单独也与文献31,32一致。调整数量和类型的细胞因子治疗，以及鸡尾酒单个细胞因子比较的能力，使学习对肾脏细胞因子暴露的具体影响该细胞培养模型的吸引?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This research was supported in part by NSF grant CHE-1229562 (VDGM), the Office of Undergraduate Research at Elon University, the Elon Chemistry Department, and the Elon Lumen Prize (TL and TAD), the Elon College Fellows Program (JAC), and the Elon College Honors Program (TAD).
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| L-glutamic acid | Sigma | G1251 | Can use the potassium salt instead. |
| malic acid | Sigma | M8304 | Can use the potassium salt instead. |
| KH2PO4 | Sigma | P0662 | |
| K2HPO4 | Sigma | P3786 | |
| EGTA | Sigma | E3889 | |
| Trisma base | Sigma | T6066 | |
| MOPS | Sigma | M3183 | |
| CCCP | Sigma | C2920 | Dilute down to 100uM as a working stock in ethanol and store at -20C. |
| Valinomycin | Sigma | V0627 | Make in DMSO and use as a 5uM working stock. Store at -20C. |
| sucrose | Fisher | S5-500 | |
| KCN | Mallinckrodt | 6379 | Make a concentrated stock in ethanol and then dilute with water |
| rotenone | Sigma | R8875 | Highly toxic. Made in ethanol. |
| oligomycin | Sigma | O4876 | Highly toxic. Made in ethanol. |
| ADP | Sigma | A2754 | |
| TMRE | Sigma | 8717-25mg | Dilute 100uM stock with EB immediatley before use. |
| DMEM | Gibco | 11965-084 | 1x regular (high glucose). |
| Pen/Strep | Invitrogen | 15140-155 | |
| L-glutamine | Fisher | SH3002101 | Store aliquots at -20C |
| FBS | Lonza | 14-501F | US origin, premium quality. Heat inactivate and store aliquots at -20 |
| Trypsin-EDTA | Sigma | T4049 | |
| DMSO | SIgma | D2650 | |
| Protien Assay Dye (5x) | Bio-Rad | 500-0006 | Any protein assay can substitute. |
| BSA | Fisher | BP1600-100 | Make 2mg/mL stock in water for protein assay. |
| MTT powder | Sigma | M2128 | Filter sterlize 5mg/mL stock made in PBS. Store aliquots at -20C; store at 4C for up to 1 week. |
| Tergitol solution (NP-40) | Sigma | NP40S | |
| Recombinant Human IL-1B | Gibco | PCH08014 | Once opened store aliquots at -20C |
| Recombinant Human TNF-alpha | Gibco | PHC3015L | |
| Recombinant Human IFN-gamma | Gibco | PHC4031 | |
| Dulbeccos PBS (-/-) | Sigma | D8537 | Make sure it is without Mg2+ and Ca2+ ions. |
| Cytochrom c ELISA kit | R&D systems | DTC0 | Human for HEK-293T cells. |
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