Name: visualizing mitophagy with fluorescent dyes for mitochondria and lysosome
Uploaded: 2022-11-30T14:30:00
Description: Watch this Scientific Journal Video about Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome at JoVE.com

这项工作由国家重点研发计划（2017YFA0105601，2018YFA0107102）、国家自然科学基金（81970333，31901044）和上海高等学校特聘教授计划（GZ2020008）部分资助。

1. 细胞培养和传代
注意：该方案以常规培养的小鼠胚胎成纤维细胞（MEF）为例进行描述。
<ol><li>在 10 cm 细胞培养皿中用 10 mL Dulbecco 改良鹰培养基 （DMEM） 培养 MEF 细胞。在37°C和5%CO2 下孵育，并在显微镜下以100倍放大倍率监测细胞。</li>
	<li>进行常规细胞传代。<ol><li>当细胞达到80%-90%汇合度（每3天）时，用2mL的Dulbecco磷酸盐缓冲盐水（DPBS）洗涤细胞。?...

<ol>
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这里描述的方案提供了一种评估和监测活细胞中线粒体自噬的动态过程的方法，包括自噬体、溶酶体和线粒体裂变，通过与细胞通透的线粒体和溶酶体染料共染色。该方法还可用于识别线粒体和评估线粒体形态。本研究中使用的两种染料都应避光，应避免多次冻融循环，并且染料应尽可能以一次性等分试样储存。建议制备染料的工作溶液，以避免将它们直接添加到细胞培养基中，这可能导致高局?...

线粒体是几乎所有真核细胞的动力源1，2。除了通过氧化磷酸化产生ATP外，线粒体在其他过程中起着至关重要的作用，例如生物能量学，钙稳态，自由基生成，细胞凋亡和细胞稳态3，4，5。当线粒体从电子传递链中的多个复合物产生活性氧（ROS）时，它们不断受到潜在氧化应激的刺激，当抗氧化防御系统崩溃时，最终会导致结构损伤和功能障碍6，7。已发现线粒体功能障碍会导致许多疾病，包括代谢紊乱、神经变性和心血管疾病8。因此，维持健康的线粒体种群及其正常功能至关重要。线粒体是高度可塑性和动态的细胞器;它们的形态和功能受线粒体质量控制机制控制，包括线粒体蛋白的翻译后修饰（PTM）、线粒体生物发生、融合、裂变和线粒体自噬9，10。由动态蛋白相关蛋白 1 （DRP1）（动态蛋白超家族的 GTP 酶）介导的线粒体裂变导致小而圆的线粒体并分离功能失调的线粒体，线粒体可以被线粒体自噬清除和降解11，12。
线粒体自噬是一种通过自噬选择性降解线粒体的细胞过程，通常发生在损伤、衰老或应激后的受损线粒体中。随后，这些线粒体被递送到溶酶体进行降解10。因此，线粒体自噬是一种分解代谢过程，有助于在多种细胞类型中保持线粒体的数量和质量处于健康状态。它在正常生理和应激条件下恢复细胞稳态中起着至关重要的作用13，14。细胞的特征在于复杂的线粒体自噬机制，其由细胞应激和发育变化的不同信号诱导。线粒体自噬调节途径分为泛素依赖性或受体依赖性15，16;泛素依赖性自噬由激酶 PINK1 和泛素连接酶 Parkin E3 募集到线粒体介导 17，18，而受体依赖性自噬涉及自噬受体与微管相关蛋白轻链 LC3 的结合，后者介导线粒体自噬以响应线粒体损伤19。
透射电子显微镜（TEM）是观察和检测线粒体自噬最常用的方法，也是最佳方法之一20。线粒体自噬的形态特征是自噬体或自噬体与溶酶体融合形成的自噬体或自溶酶体，这可以从电子显微镜图像中观察到21。然而，电子显微镜（EM）的弱点是无法监测线粒体自噬的动态过程，例如线粒体去极化，线粒体裂变以及自噬体和溶酶体的融合，在活细胞20中。因此，通过成像活细胞器评估线粒体自噬是线粒体研究的一种有吸引力的替代方法。这里描述的活细胞成像技术使用两种荧光染料对线粒体和溶酶体进行染色。当线粒体自噬发生时，被自噬体吞噬的受损或多余的线粒体被线粒体染料染成绿色，而红色染料会染色溶酶体。这些自噬体和溶酶体的融合，称为自溶酶体，导致绿色和红色荧光重叠并表现为黄点，从而表明线粒体自噬的发生22。细胞通透的线粒体染料（MitoTracker Green）含有轻度硫醇反应性氯甲基部分，用于标记线粒体23。为了标记线粒体，只需将细胞与染料一起孵育，染料被动扩散到质膜上并积聚在活性线粒体中。这种线粒体染料很容易染色活细胞，并且在染色醛固定或死细胞方面效果较差。溶酶体染料（LysoTracker Red）是一种荧光嗜酸探针，用于标记和跟踪活细胞中的酸性细胞器。该染料对酸性细胞器表现出高选择性，并且可以有效地标记纳摩尔浓度的活细胞24。
本文介绍了在活细胞中使用这些荧光染料的程序，包括装载染料以及线粒体和溶酶体的可视化。这种方法可以帮助研究人员使用活细胞荧光显微镜观察线粒体自噬。它还可用于量化线粒体和溶酶体，并评估线粒体形态。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Automated cell counter</td>
			<td>Countstar</td>
			<td>IC1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counting chamber slides</td>
			<td>Countstar</td>
			<td>12-0005-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle medium (DMEM)</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td>Corning</td>
			<td>21-031-CVC</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom cell culture dish (confocal dish)</td>
			<td>NEST</td>
			<td>801002</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J (Rasband, NIH)</td>
			<td>NIH</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/download.html</td>
		</tr>
		<tr>
			<td>Krebs&#8211;Henseleit(KHB) buffer</td>
			<td></td>
			<td></td>
			<td>Self-prepared</td>
		</tr>
		<tr>
			<td>LysoTracker Red</td>
			<td>Invitrogen</td>
			<td>1818430</td>
			<td>100 &#181;mol/L, red-fluorescent lysosome dye</td>
		</tr>
		<tr>
			<td>MitoTracker Green</td>
			<td>Invitrogen</td>
			<td>1842298</td>
			<td>200 &#181;mol/L stock, green-fluorescent mitochondria dye</td>
		</tr>
		<tr>
			<td>Mouse Embryonic Fibroblasts</td>
			<td></td>
			<td></td>
			<td>Self-prepared</td>
		</tr>
		<tr>
			<td>Objective (63x oil lens)</td>
			<td>ZEISS</td>
			<td>ZEISS LSM 880</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25%</td>
			<td>Gibico</td>
			<td>Cat# 25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEISS LSM 880 Confocal Laser Scanning Microscope</td>
			<td>ZEISS</td>
			<td>ZEISS LSM 880</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEN Microscopy Software 2.1 (confocal microscope imaging software)</td>
			<td>ZEISS</td>
			<td>ZEN 2.1</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing mitophagy with fluorescent dyes for mitochondria and lysosome

线粒体是细胞的动力源，在生物能量学、自由基生成、钙稳态和细胞凋亡中起着重要作用。线粒体自噬是线粒体质量控制的主要机制，通常使用显微镜观察进行研究，但 体内 线粒体自噬测定难以进行。通过对活细胞器进行成像来评估线粒体自噬是线粒体研究的另一种必要方法。该协议描述了在活细胞中使用细胞通透性绿色荧光线粒体染料MitoTracker Green和红色荧光溶酶体染料LysoTracker Red的程序，包括染料的负载，线粒体和溶酶体的可视化，以及预期结果。还提供了评估活细胞线粒体自噬的详细步骤，以及有关显微镜软件设置的技术说明。这种方法可以帮助研究人员使用活细胞荧光显微镜观察线粒体自噬。此外，它还可用于量化线粒体和溶酶体并评估线粒体形态。

MitoTracker Green是一种绿色荧光线粒体染色剂，能够准确地定位为线粒体。该染料很容易染色活细胞，并且在染色醛固定细胞或死细胞方面效果较差（图2）。红色荧光溶酶体染料LysoTracker Red能够标记和跟踪酸性溶酶体细胞器，并且只能对活细胞进行染色（图2）。共聚焦显微镜成像允许可视化用适当染料染色的线粒体和溶酶体（图1 ?...

Watch this Scientific Journal Video about Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome at JoVE.com

Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome

线粒体自噬是线粒体质量控制的主要机制。然而， 由于 缺乏可靠的定量测定，体内线粒体自噬的评估受到阻碍。这里介绍的是使用细胞通透性绿色荧光线粒体染料和红色荧光溶酶体染料观察活细胞中线粒体自噬的方案。

用荧光染料可视化线粒体和溶酶体的线粒体自噬

Mitochondria, being the powerhouses of the cell, play important roles in bioenergetics, free radical generation, calcium homeostasis, and apoptosis. ...

该方法有助于使用细胞通透性绿色荧光线粒体染料和红色荧光溶酶体染料观察活细胞中的线粒体自噬。线粒体自噬在维持线粒体稳态和细胞功能的各个方面起着重要作用。该技术可以为治疗线粒体相关疾病提供一种新的方法。此过程不是很复杂。捕获清晰的图像对于计数线粒体和溶酶体的叠加非常重要。首先，在装有10毫升Dulbecco改良鹰培养基或DMEM的10厘米细胞培养皿中培养小鼠胚胎成纤维细胞或MEF细胞。在 37 摄氏度和 5% 二氧化碳下孵育，并在显微镜下以 100X 放大倍率监测细胞。当细胞达到80%至90%汇合时，用两毫升Dulbecco的磷酸盐缓冲盐水洗涤它们。然后加入两毫升0.05%胰蛋白酶EDTA一分钟以解离细胞，然后加入两毫升DMEM以停止反应。将细胞悬液以 100 G 离心三分钟，然后将沉淀重悬于一毫升 DMEM 中。使用自动细胞计数器和细胞计数室载玻片对细胞进行计数，并将1.5乘以10至第六个细胞接种到含有10毫升DMEM的新10厘米细胞培养皿中。对于线粒体自噬测定，如前所述制备细胞悬液，并将细胞悬液稀释至每毫升新鲜DMEM的10至第五个细胞的1倍。将两毫升稀释的细胞悬液加入20毫米共聚焦培养皿中，并交叉摇动培养皿。在 37 摄氏度和 5% 二氧化碳下孵育 24 小时。通过稀释储备溶液来制备染料的工作溶液。向2毫升DMEM中加入0.2毫摩尔线粒体染料和50微摩尔溶酶体染料各2微升，得到工作浓度为0.2微摩尔线粒体染料和50纳摩尔溶酶体染料。从共聚焦培养皿中取出培养基，并加入一毫升染色溶液以覆盖细胞。将细胞培养皿置于37摄氏度和5%二氧化碳下20至30分钟。设置共聚焦显微镜成像软件的参数。对于双激发图像，使用488纳米和543纳米的顺序激发，并分别收集505至545纳米和大于560纳米的发射。从培养箱中取出含有染料的培养基培养皿，并向培养皿中加入一毫升克雷布斯-亨塞莱特或KH缓冲液。为了诱导线粒体自噬，在室温下用KH缓冲液中的一微摩尔FCCP处理细胞10分钟，并立即使用共聚焦显微镜对细胞进行成像。在 63X 油性镜头顶部涂抹适量的油。将样品放在共聚焦显微镜的样品台上，并将其直接移动到物镜上方。使用成像软件通过单击软件界面左上角的定位选项卡来查找样品。为实验选择绿色过滤器集。使用粗调旋钮通过上下移动物镜来快速对焦。通过目镜清晰可见细胞样本后，搜索并聚焦单个细胞的区域，并将其移动到视野的中心。单击软件界面左上角的采集选项卡以获取图像。仅选择 488 纳米通道和帧分辨率 1024 x 1024 进行预览。单击左上角的实时选项卡以开始实时扫描。将视野调整到最清晰，并通过向左或向右移动滑块来调整激光功率。将增益设置保持在 600 以下以避免过度曝光。将针孔值调整为 156，将增益值调整为 545，将数字偏移值调整为零。选择最佳视野，检查两个通道并选择帧分辨率 1024 x 1024。单击捕捉以获取 2D 图像并保存获取的图像。在这项研究中，FFCP用于触发MEF细胞中的线粒体自噬以进行共聚焦成像。此处显示了用显示线粒体的绿色荧光线粒体染料染色的细胞和用显示溶酶体的红色荧光溶酶体染料染色的细胞的代表性图像。当受损的绿色染色线粒体被红色染色的溶酶体吞噬时，绿色和红色荧光重叠以显示黄色共定位线粒体溶酶体。黄点对应于这些代表正在进行的线粒体自噬的共定位线粒体溶酶体，因此可以计数以评估线粒体自噬的程度。制备用于线粒体和溶酶体染色的染料工作溶液以及在用共聚焦显微镜成像时保持适当的细胞汇合度是要记住的关键步骤。该程序还可用于分析线粒体数量和形态，这对于评估线粒体动力学很重要。线粒体自噬与多种人类疾病密切相关，该技术可用于探索线粒体自噬与人类疾病之间的联系。

Research

JoVE Journal

Biology

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

与非侵入性检测荧光染料标记的干细胞与光学成像

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

科研

生物学

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

可视化的Live果蝇胶质细胞神经​​肌肉交界处，与荧光染料

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

可视化的单分子荧光显微镜下的DNA复制

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

使用荧光显微镜可视化中的细菌线虫

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

单线粒体超氧闪烁在完整心脏或共聚焦成像<em&gt;在体内</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

指南现代荧光定量免疫印迹与故障排除策略

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

遗传条形码与荧光蛋白的复用的应用程序

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Visualizing Stromule Frequency with Fluorescence Microscopy

可视化Stromule频率与荧光显微镜

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

pH 依赖性荧光 Keima 对流式细胞术 Mitophagy 的敏感性测定

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between ...

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

膜和细胞壁选择性荧光染料在丝状真菌活细胞成像中的应用

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two ...