我们感谢埃文斯实验室的成员对这份手稿的深思熟虑的反馈。这项工作得到了杜克怀特黑德学者，杜克科学和技术学者和霍华德休斯医学研究所（HHMI）汉娜格雷奖学金的支持。 图1A 是使用 BioRender.com 制作的。

1. 生物样品的制备
注意：在生物安全柜中使用无菌技术执行以下步骤。用70%乙醇喷洒机柜表面和所有材料。
<ol><li>HeLa细胞培养和转染<ol><li>在Dulbecco的改良鹰培养基（DMEM）中培养30，000个HeLa细胞，含有4.5g / L葡萄糖，补充有10%胎牛血清和1%L-谷氨酰胺溶液（HeLa培养基;参见 材料表）。将细胞铺在含有 2 mL 预热 HeLa 培养基的 35 mm 玻璃底成像皿（参见<str...

线粒体膜电位和超氧化物水平的定量

此处概述的工作流程可用于使用基于荧光的成像稳定且可重复地量化线粒体膜电位和超氧化物水平30。在设计这些实验时，需要考虑重要的技术限制。用空的YFP载体YFP-ParkinWT或YFP-ParkinT240R转染HeLa细胞。空的YFP载体被用作对照，以确认实验结果是帕金特有的。对于瞬时转染，通过实验确定DNA与转染试剂的质量比为1：3，以产生最高的转染率，其中每个构建体使用2 μ...

线粒体网络是一系列相互连接的细胞器，在能量产生1，先天免疫2，3和细胞信号传导4，5中起着至关重要的作用。线粒体失调与帕金森病（PD）等神经退行性疾病有关6，7。PD是一种进行性神经退行性疾病，影响黑质的多巴胺能神经元，影响全球近1000万人8。PD与线粒体自噬有遗传联系，线粒体自噬是维持细胞稳态所必需的线粒体质量控制途径，选择性地去除受损的线粒体9，10。研究已经确定了多种独立的线粒体自噬途径，包括含有 1 （FUNDC1） 介导的线粒体自噬的 FUN14 结构域、Bcl-2 相互作用蛋白 3 （BNIP3） 促进的线粒体自噬、NIX 依赖性线粒体自噬以及特征明确的 PTEN 诱导激酶 1 （PINK1）/帕金调节线粒体自噬10，11。PINK1（一种假定的激酶）和帕金（一种E3泛素连接酶）与磷酸泛素受损线粒体协同工作，后者驱动自噬体的形成，吞噬受损细胞器并与溶酶体融合以启动降解12，13，14，15，16。帕金的突变与PD相关的表型有关，例如通过多巴胺能神经元丢失引起的神经变性17，18。
在这里，描述了一个协议，其中HeLa细胞，常规使用的源自宫颈癌的永生化细胞，用于研究Parkin在维持线粒体网络健康中的作用。HeLa细胞表达的内源性帕金水平可以忽略不计，因此需要外源性帕金表达19。为了研究Parkin在线粒体网络健康中的作用，用野生型Parkin（ParkinWT），Parkin突变体（ParkinT240R）或空对照载体转染HeLa细胞。ParkinT240R是一种常染色体隐性幼年帕金森综合征突变，影响Parkin E3连接酶活性，显着降低线粒体自噬途径的效率20。HeLa细胞暴露于轻度（5μM）或严重（20μM）浓度的羰基氰间氯苯基腙（CCCP），一种线粒体解偶联剂。用高浓度的CCCP处理通常用于在各种细胞系中诱导帕金介导的线粒体自噬，例如HeLa和COS-7细胞21，22，23。
治疗后，该方案使用两种当前可用的线粒体靶向荧光染料对线粒体网络进行实时成像。四甲基罗丹明、乙酯、高氯酸盐 （TMRE） 是一种阳离子染料，其荧光基于线粒体膜电位24，而 MitoSOX 是一种线粒体超氧化物指示剂，其中荧光强度是超氧化物浓度25 的函数。最后，概述的方案使用基于荧光的定量和简单的工作流程来有效地量化线粒体膜电位和超氧化物水平，而用户偏差的范围最小。尽管该协议旨在研究HeLa细胞中的线粒体功能，但它可以适用于其他细胞系和原代细胞类型，以定量表征线粒体网络健康。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals, Peptides, and Recombinant Proteins</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CCCP (carbonyl cyanide m-chlorophenyl hydrazone)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C2759</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (1x) with 4.5 g/L glucose</td>
			<td>Gibco</td>
			<td>11-965-084</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO, Anhydrous</td>
			<td>ThermoFisher Scientific</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH3007103</td>
			<td></td>
		</tr>
		<tr>
			<td>FuGENE 6 (Tranfection Reagent)</td>
			<td>Promega</td>
			<td>E2691</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX 100x (L-Glutamine Solution)&#160;</td>
			<td>Gibco&#160;</td>
			<td>35-050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoescht 33342</td>
			<td>ThermoFisher Scientific</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoSOX&#160; Red&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>M36008</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker Deep Red</td>
			<td>ThermoFisher Scientific</td>
			<td>M7514</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM (Redued Serum media)</td>
			<td>ThermoFisher scientific</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE)&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>T669</td>
			<td></td>
		</tr>
		<tr>
			<td>Experimental models: Organisms/Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa-M (Homo sapiens)</td>
			<td>A. Peden (Cambridge Institute for Medical Research)</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant DNA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EYFP Empty Vector</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>YFP-Parkin T240R</td>
			<td>This Paper</td>
			<td>Generated by site-directed mutagenesis from YFP-Parkin</td>
			<td></td>
		</tr>
		<tr>
			<td>YFP-Parkin WT</td>
			<td>Addgene; PMID:19029340</td>
			<td>RRID:Addgene_23955</td>
			<td></td>
		</tr>
		<tr>
			<td>Software and Algorithms</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adobe Illustrator</td>
			<td>Adobe Inc.</td>
			<td>https://www.adobe.com/products/illustrator</td>
			<td>(Schindelin, 2012)</td>
		</tr>
		<tr>
			<td>Excel (Spreadsheet Software)</td>
			<td>Microsoft Office&#160;</td>
			<td>https://www.microsoft.com/en-us/microsoft-365/excel</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td></td>
			<td>https://imagej.net/software/fiji/</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Application Suite (LAS X)</td>
			<td>Leica</td>
			<td>https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft Office</td>
			<td>https://www.microsoft.com/excel</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism9 (Statistical Analysis Software)</td>
			<td>GraphPad Software</td>
			<td>https://www.graphpad.com</td>
			<td></td>
		</tr>
		<tr>
			<td>Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Dish, No. 1.5 Coverslip, 20 mm Glass Diameter, Uncoated</td>
			<td>MatTek</td>
			<td>P35G-1.5-20-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Cage Incubator (Environmental Chamber)</td>
			<td>Okolab</td>
			<td>https://www.oko-lab.com/cage-incubator</td>
			<td></td>
		</tr>
		<tr>
			<td>DMiL Inverted Microscope</td>
			<td>Leica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>LIGHTNING Deconvolution Software</td>
			<td>Leica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>STELLARIS 8 confocal microscope</td>
			<td>Leica</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescence-based quantification of mitochondrial membrane potential and superoxide levels using live imaging in hela cells

线粒体是通过ATP合成 控制 能量产生，对代谢稳态至关重要的动态细胞器。为了支持细胞代谢，各种线粒体质量控制机制合作以维持健康的线粒体网络。其中一种途径是线粒体自噬，其中PTEN诱导的激酶1（PINK1）和受损线粒体的Parkin磷酸化泛素化促进自噬体隔离并随后通过溶酶体融合 从 细胞中去除。线粒体自噬对细胞稳态很重要，帕金的突变与帕金森病（PD）有关。由于这些发现，人们非常重视研究线粒体损伤和周转，以了解线粒体质量控制的分子机制和动力学。在这里，活细胞成像用于可视化HeLa细胞的线粒体网络，以量化线粒体解偶联剂羰基氰间氯苯基腙（CCCP）处理后的线粒体膜电位和超氧化物水平。此外，表达抑制帕金依赖性线粒体自噬的帕金PD连锁突变（ParkinT240R）以确定与表达野生型帕金的细胞相比突变如何影响线粒体网络。此处概述的方案描述了一种简单的工作流程，使用基于荧光的方法有效地量化线粒体膜电位和超氧化物水平。

The mitochondrial network is a series of interconnected organelles that play a crucial role in energy production1, innate immunity2,3, and cell signalling4,5. Mitochondrial dysregulation has been associated with neurodegenerative diseases such as Parkinson&#39;s disease (PD)6,7. PD is a progressive neurodegenerative disorder affecting dopaminergic neurons of the substantia nigra that impacts nearly 10 million people worldwide8. PD has been genetically linked to mitophagy, a mitochondrial quality control pathway necessary for maintaining cellular homeostasis that selectively removes damaged mitochondria9,10. Studies have identified multiple independent mitophagy pathways, including FUN14 domain containing 1 (FUNDC1)-mediated mitophagy, Bcl-2 interacting protein 3 (BNIP3)-facilitated mitophagy, NIX-dependent mitophagy, and the well-characterized PTEN-induced kinase 1 (PINK1)/Parkin-regulated mitophagy10,11. PINK1 (a putative kinase) and Parkin (an E3 ubiquitin ligase) work in tandem to phospho-ubiquitinate damaged mitochondria, which drives the formation of autophagosomes that engulf the damaged organelle and fuse with lysosomes to initiate degradation12,13,14,15,16. Mutations in Parkin have been associated with PD-linked phenotypes such as neurodegeneration via the loss of dopaminergic neurons17,18.
Here, a protocol is described in which HeLa cells, routinely used immortalized cells derived from cervical cancer, are used to investigate the role of Parkin in maintaining mitochondrial network health. HeLa cells express negligible levels of endogenous Parkin and therefore require exogenous Parkin expression19. To study the role of Parkin in mitochondrial network health, HeLa cells are transfected with either wild-type Parkin (ParkinWT), a Parkin mutant (ParkinT240R), or an empty control vector. ParkinT240R is an autosomal recessive juvenile parkinsonism mutation that affects Parkin E3 ligase activity, significantly reducing the efficiency of the mitophagy pathway20. HeLa cells are subject to mild (5 &#181;M) or severe (20 &#181;M) concentrations of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent. Treatment with severe concentrations of CCCP is routinely used to induce Parkin-mediated mitophagy in various cell lines, such as HeLa and COS-7 cells21,22,23.
Following treatment, the protocol employs live imaging of the mitochondrial network using two currently available mitochondrial-targeted fluorescent dyes. Tetramethylrhodamine, ethyl ester, perchlorate (TMRE) is a cationic dye that fluoresces based on mitochondrial membrane potential24, while MitoSOX is a mitochondrial superoxide indicator where the fluorescence intensity is a function of superoxide concentration25. Finally, the outlined protocol uses a fluorescence-based quantification and simple workflow to effectively quantify the mitochondrial membrane potential and superoxide levels with minimal scope for user bias. Although this protocol was designed to study mitochondrial function in HeLa cells, it can be adapted for additional cell lines and primary cell types to quantitively characterize mitochondrial network health.

作者聚焦：在 HeLa 细胞中使用实时成像对线粒体膜电位和超氧化物水平进行基于荧光的定量  

使用HeLa细胞中的实时成像对线粒体膜电位和超氧化物水平进行基于荧光的定量

This research focuses on mitochondria dysregulation in neurodegenerative diseases. We believe that this research can be used to understand potential causes for disease onset that could lead to therapeutics combating neurodegenerative diseases like Parkinson's disease and ALS. Techniques such as super resolution microscopy like STED and SIM or expansion microscopy have improved the ability to accurately understand protein distribution within individual organelles and mitochondria distribution throughout the cell.Results of this technique can be used as a starting point to study the effect of Parkinson's disease linked mutations on mitochondrial turnover and begin to understand the importance of regulating reactive oxygen species levels and mitochondrial membrane potential to maintain neuronal health. Our laboratory aims to mechanistically characterize individual mitochondrial quality control pathways to understand the interplay between these pathways. By having insights into pathway dynamics, one can understand how mitochondria are maintained and how mitochondrial dysregulation contributes to neurodegenerative disease onset.

在该协议中，基于荧光的定量用于测量CCCP处理后线粒体网络的膜电位和超氧化物水平（图1）。该工作流程使用了HeLa细胞，这是一种源自宫颈癌的永生化细胞系。HeLa细胞通常用于研究线粒体生物学，并且相对平坦，因此很容易使用显微镜可视化线粒体网络。为了研究Parkin在维持线粒体网络健康中的作用，用空对照载体ParkinWT或ParkinT240R （一种破坏线粒体自噬...

Watch this Scientific Journal Video about Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells at JoVE.com

该技术描述了一种有效的工作流程，使用基于荧光的实时成像可视化和定量测量HeLa细胞内的线粒体膜电位和超氧化物水平。

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, ...

本研究的重点是神经退行性疾病中的线粒体失调。我们相信，这项研究可用于了解疾病发作的潜在原因，这些原因可能导致对抗帕金森病和ALS等神经退行性疾病的治疗方法。STED和SIM等超分辨率显微镜或扩增显微镜等技术提高了准确了解单个细胞器内的蛋白质分布和整个细胞线粒体分布的能力。该技术的结果可以作为研究帕金森病相关突变对线粒体周转影响的起点，并开始了解调节活性氧水平和线粒体膜电位对维持神经元健康的重要性。我们的实验室旨在机械地表征单个线粒体质量控制途径，以了解这些途径之间的相互作用。通过深入了解通路动力学，人们可以了解线粒体如何维持以及线粒体失调如何导致神经退行性疾病的发作。

使用HeLa细胞中的实时成像对线粒体膜电位和超氧化物水平进行基于荧光的定量

Abstract