Name: a flow cytometry-based assay for measuring mitochondrial membrane potential in cardiac myocytes after hypoxia/reoxygenation
Uploaded: 2018-07-13T13:00:00.000+00:00
Duration: 7 min 14 s
Description: Watch this Scientific Journal Video about A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation at JoVE.com

本研究由中国国家重点研究开发项目 (2017YFC1700503)、全国基础研究计划 (973 计划)、中国国家自然科学基金 (81370223 号) 资助。81573957号), 以及北京协和医学院研究生创新研究基金会 (2016-1002-01-02)。

1. 试剂和溶液的制备
<ol><li>根据制造商的指示, 加入25毫升肌细胞生长培养基补充剂, 以500毫升的细胞生长培养基, 制备人心肌肌 (母国) 全培养基。在使用前贮存在4°c 和温暖到37°c。</li>
	<li>将 txl-特格尔超细粉溶于血清/葡萄糖 Dulbecco 修饰鹰的培养基 (DMEM) 中, 制备通心络 (txl-特格尔) 溶液, 并通过添加 txl-特格尔将µg 浓度调整为 400 DMEM/毫升 (详情见11)。在使用前贮存在4°c 和温暖到37°c。</li>
	<li>在黑暗中, 在15毫升离心管中加入25µL JC-1 溶液 (200x)、4毫升超纯水和1毫升的库存染色缓冲器 (5x), 准备5毫升的 JC-1 工作溶液。在使用前用吸管将混合物上下数次, 加热到37摄氏度。</li>
	<li>在50毫升离心管中加入24毫升的超纯水到6毫升的库存染色缓冲器 (5x), 制备30毫升工作染色缓冲器。使用此解决方案冰冷。</li>
</ol>2. 建立缺氧/复氧模型
<ol><li>板 1.0 x 105细胞每井与 HCM 完全媒介在6个井板材。生长达到约70% 的融合在细胞孵化器包含 5% CO2和95% 空气在37°c。</li>
	<li>用磷酸盐缓冲盐水 (PBS) 清洗母国措施。使他们接触不同的治疗方法 (400 µg/毫升 txl-特格尔或不) 在2毫升血清/葡萄糖无 DMEM 30 分钟缺氧前的细胞孵化器包含 5% CO2和95% 空气37°c。</li>
	<li>在密闭和缺氧的罐子 (2.5 升) 上孵化母国措施, 装有催化剂来清除游离氧, 并诱导18小时缺氧 (图 1A), 以及用于测量介质11、12中氧张力的厌氧指示器,13, 在一个细胞孵化器包含 5% CO2和95% 空气在37°c。 注: 催化剂在罐中清除自由氧需要大约1.5 小时。因此, 在罐子打开之前, 母国措施在罐子里保持19.5 小时。在缺氧条件下, 厌氧指示器的颜色由淡蓝色变化为常氧白色 (图 1B)。</li>
	<li>Reoxygenate 母国措施将6井板块移动到一个孵化器设置在37°c, 包含 5% CO2和95% 空气 2 h。</li>
</ol>3. 流式细胞术用母国措施的研制
<ol><li>将0.25% 胰蛋白酶-乙二胺四乙酸 (EDTA) 溶液和 PBS 加热至37摄氏度。</li>
	<li>从各井中抽出培养基, 用1毫升 PBS 冲洗细胞, 并在井壁上加0.5 毫升0.25% 胰蛋白酶。</li>
	<li>孵化37摄氏度, 直到所有的母国措施几乎分离 (约1分钟)。</li>
	<li>添加1毫升的 DMEM 完全培养基 (DMEM 培养基和10% 胎牛血清) 到井中中和胰蛋白酶。在室温下将细胞悬浮液转移到15毫升离心管和颗粒离心, 在 500 x g 处进行3分钟。</li>
	<li>小心地吸入上清液而不干扰颗粒。</li>
	<li>添加0.5 毫升的 HCM 完整培养基和0.5 毫升的 JC-1 工作溶液, 每管。并用重悬吹打。</li>
	<li>用铝箔覆盖整个管子, 以防止荧光指示器的漂白和在正常情况下 (在37摄氏度含 5% CO2和95% 空气中) 的情况下在20分钟内孵化母国措施。</li>
	<li>颗粒母国措施由离心在 500 x g 3 分钟在4°c。</li>
	<li>用2毫升冰冷 JC-1 染色缓冲器冲洗两次母国措施。</li>
	<li>在样品管 (12 毫米 x 75 毫米) 的最后一卷300µL 的冰冷染色缓冲器中并用重悬母国措施, 并在30分钟内使用细胞进行分析。 注: 使用羰基氰化物 m-氯苯基腙 (CCCP), 一种有效的呼吸电子转运链抑制剂, 治疗1小时的母国措施, 浓度为20µM, 为阳性对照。</li>
</ol>4. 流式细胞仪设置
<ol><li>启动流式细胞仪, 并确保软件连接到它。</li>
	<li>执行射流技术启动, 启动流并设置 breakoff。</li>
	<li>在流式细胞术软件中打开两个点绘制窗口。<ol>
<li>选择在第一个点图中 Y 轴上的 X 轴和侧散射光 (SSC) 上的正散射光 (FSC), 分别表示单元格的大小和粒度。</li>
		<li>使用第二点图中的对数刻度为 X 轴选择 PE (575 英寸 26 nm) 检测通道, 用于测量 FITC 中的 JC-1 染料的荧光强度。</li>
	</ol>
</li>
</ol>5. 流式细胞仪测量和数据分析
<ol><li>单击 "卸载", 释放管道支撑臂并定位空白(未用 JC-1 染色的母国措施)。单击 "加载", 将管道支撑臂返回到其工作位置。</li>
	<li>单击 "记录" 从空取样管中的悬浮中收集粒子, 然后在第一个点图中对单元格填充 (P1) 进行进一步分析 (图 3)。</li>
	<li>收集其他取样管的母国措施, 通过调整荧光通道的电压来优化测量, 以确保单元格点位于第二点图的中心区域。</li>
	<li>通过将红荧光强度的比值除以绿色荧光强度, 显示各样本管的统计值, 并计算出每个样品的基质金属蛋白酶。</li>
</ol>

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作者没有什么可透露的。

在这里, 我们提出一个协议, 使用 JC-1 染料来评估细胞的基质干细胞暴露后, 通过 JC-1 检测发现, 细胞的基质干细胞是独立的因素, 如线粒体大小, 形状和密度可能影响单组分荧光信号14。因此, JC-1 化验结果比较可靠。同时, 对 JC-1 的检测具有很方便、省时的性能。这种检测对材料和试剂的要求很低, 一般情况下, 从细胞分离到基质金属蛋白酶的测量, 其染色需要少于1.5 小时。
如果研究人员想要获得可靠和满意的结果, 那么在执行 JC-1 分析时, 一些关键步骤是不能被夸大的。首先, 装满 JC-1 的细胞应储存在冰冷的染色缓冲中, 直到它们被流式室采集。温度对基质金属蛋白酶的稳定性有很大影响, 在室温下, 一个样品的值在短时间内变化很大。此外, 还建议初步实验确定 CCCP 的预处理时间和浓度, 确保阳性对照的红/绿比为0。值得注意的是, 羰基氰化物 4-(三氟甲氧基) 苯腙 (FCCP), 这也可以消除基质金属蛋白酶和抑制氧化磷酸, 是一种替代 CCCP, 也可以用来建立积极的控制10。
JC-1 检测有一个主要的限制, 因为其他荧光不能与 JC-1 结合, 因为它的荧光发射跨越绿色, 黄色, 和部分的红色波长的光谱。此外, JC-1 化验必须结合一个或多个方法, 如使用 calcein-acetoxymethylester15,16 , 甚至基因操作17,18, 以确定是否 mPTP 的开放帐户基质金属蛋白酶的减少。这是因为开放其他线粒体毛孔像 ATP 敏感的钾离子通道19,20也可以导致线粒体的退极化。
JC-1 分析最适合于更粗的 "是/否" 评估, 目的是评估线粒体是否主要是极化的 (例如,与凋亡相关的实验)8。它也被用于测量不同细胞类型的基质干细胞, 包括神经元21, 肝细胞22, 肾细胞23 , 甚至孤立的线粒体24。总之, 我们描述了一个快速, 简单和灵敏的方法, 以检测在母国措施后, 对中的基质金属蛋白酶。

冠心病是全世界死亡的主要原因。ST 段抬高心肌梗死患者减少缺血性损伤和限制梗死大小的选择是通过经皮冠状动脉介入治疗 (PCI) 的及时有效的心肌再灌注1, 2。然而, 再灌注导致额外的损伤, 可能占到最后梗塞大小的30% 的3。普遍认为线粒体通透性过渡孔隙 (mPTP) 不仅是缺血/再灌注过程中线粒体损伤和细胞死亡的中枢, 而且也是心肌梗死信号4的收敛目标。,5. 随着 mPTP 的开放将导致内线粒体膜电位的退极化 (基质金属蛋白酶)4, 我们检测到 mPTP 开放使用 5,5′, 6,6′四氯化碳 1,1′, 3,3′-四乙基 imidacarbocyanineiodide (JC-1) 化验。
JC-1 法是一种定性和定量的 cytofluorimetric 方法, 通过对单个线粒体6级的基质细胞进行分析, 进一步验证了它的有效性。JC-1 作为一种聚集形式存在, 在线粒体基质中产生红色到橙色的辐射 (590 17.5 nm);随着基质的丧失, JC-1 被转化为单体形式, 产生绿色荧光, 发射量为 530-15 nm。因此, 红/绿荧光强度比的降低可以表明在缺血/再灌注 (I/R) 条件下基质金属蛋白酶的减少。
除了 JC-1, 还研究了膜渗透亲脂阳离子, 如罗丹明123和 3,3′-dihexyloxadicarbocyanine 碘 [DiOC6 (3)]。然而, 与这两种探头相比, JC-1 更能可靠地分析基质金属蛋白酶。罗丹明123具有相对较差的灵敏度 (特别是在淬火方式7,8) 和较差的特异性。在罗丹明123的转变有时是如此之小, 研究人员或设备很难观察/检测。此外, 在单个细胞中, 罗丹明123有不同的线粒体结合点, 因此它可能有不同的荧光排放量9。DiOC6 (3) 不推荐用于检测基质金属蛋白酶, 因为它对等离子体膜10的退极化反应灵敏。
因此, 在这里, 我们使用 JC-1 的方法来评估在接触到缺氧/复氧或没有保护剂的情况下母国措施的基质。

<table><tbody><tr><td>Mitochondrial membrane potential assay kit with JC-1</td><td>Beyodtime, China</td><td>C2006</td><td>In the kit there are JC-1 stock solution (200&amp;times;), stock staining buffer (5&amp;times;) and CCCP(10mM)</td></tr><tr><td>Tongxinluo ultrafine powder</td><td>Shijiazhuang Yiling Pharmaceutical Co., China</td><td>071201</td><td></td></tr><tr><td>Annexin V-FITC/PI Kit</td><td>Becton-Dickinson, USA</td><td>556547</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies, Grand Island Biological Company, USA</td><td>11966-025</td><td></td></tr><tr><td>Human cardiac myocyte</td><td>Promocell, Germany</td><td>C-12810</td><td></td></tr><tr><td>Myocyte Growth Medium&lt;br/&gt; (SupplementMix)</td><td>Promocell, Germany</td><td>C-39275</td><td></td></tr><tr><td>Myocyte Growth Medium (Ready-to-use)</td><td>Promocell, Germany</td><td>C-22070</td><td>used with Myocyte Growth Medium SupplementMix</td></tr><tr><td>GENbox</td><td>BioM&amp;eacute;rieux, Marcy l&amp;rsquo;Etoile, France</td><td>96127</td><td>2.5L</td></tr><tr><td>Catalyst (AnaeroPack)</td><td>MITSUBISHI GAS CHEMICAL&amp;nbsp;COMPANY, INC.&amp;nbsp;, Japan</td><td>&amp;nbsp;C-1</td><td></td></tr><tr><td>Anaerobic indicator</td><td>BioM&amp;eacute;rieux, Marcy l&amp;rsquo;Etoile, France</td><td>96118</td><td></td></tr><tr><td>Flow cytometer</td><td>Becton-Dickinson, USA</td><td>FACSAria 2</td><td></td></tr><tr><td>BD FACSDiva Software</td><td>Becton-Dickinson, USA</td><td></td><td>Version8.0.1</td></tr><tr><td>Sample tube</td><td>Corning science, USA</td><td>352054</td><td>12*75mm</td></tr><tr><td>PBS</td><td>Hyclone, USA</td><td>SH30256.01</td><td></td></tr></tbody></table>

a flow cytometry-based assay for measuring mitochondrial membrane potential in cardiac myocytes after hypoxia/reoxygenation

及时有效地再灌注闭塞性冠状动脉是降低 st 段抬高心肌梗死患者心肌梗死大小的最佳策略。然而, 再灌注本身可能导致进一步的心肌细胞死亡, 这一现象被称为再灌注损伤。线粒体通透性过渡孔隙 (mPTP) 的开放, 随着线粒体膜电位 (基质) 的减少, 或线粒体的退极化, 被普遍认为是再灌注损伤的最后一步, 负责线粒体和心肌细胞死亡。JC-1 是一种脂溶性阳离子染料, 在线粒体中积累取决于基质的价值。基质金属蛋白酶越高, 线粒体中的 JC-1 积累越多。线粒体中 JC-1 含量的增加可以通过荧光发射从绿色 (~ 530 nm) 到红色 (~ 590 nm) 来反映出来。因此, 降低红/绿荧光强度比可以表明线粒体的退极化。在这里, 我们利用 JC-1 测量基质金属蛋白酶, 或在缺氧/复氧后的人心肌细胞中 mPTP 的开放, 通过流式细胞术检测。

在进行 JC-1 试验以评估基质金属蛋白酶的变化之前, 强烈建议进行实验以确认研究人员成功确定的条件。如流式细胞术结果 (图 2) 所示, 与正常组相比, 缺氧/复氧 (H/r) 显著诱导了母国措施 (蛋白 V +/PI±) 的凋亡, 表明我们建立了一个基于细胞的 I/R (45.00 2.13% vs) 模型。11.50, 0.18% 在正常组, p < 0.05)。通心络是一种具有心肌作用的中药, 在 h/r 组 ( p < 0.05) 的条件下, 防止母国措施的凋亡 (24.50 @ 1.13% vs 45.00 @ 2.13%)。
CCCP 是一种 protonophore, 它能抑制线粒体中的氧化磷酸化, 并将电子转运链中电子载体正常活动时建立的质子梯度 (基质损耗) 解耦。因此, 用 CCCP 处理的母国措施被设置为积极控制。如图 4所示, CCCP 治疗组的红/绿比值几乎为零。与正常组相比, h-/R 治疗组显示的红/绿比值明显低于正常值 (0.69 0.07 vs 5.64 @ 0.21, 正常组为p < 0.05), 通心络逆转此比值。变化 (2.92 0.22 vs 0.69 @ 0.07 在 H/R 组, p< 0.05)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57725/57725fig1.jpg"> 图1。建立缺氧/复氧 (h-/R) 模型.a. 建立 H/R 模型所需的材料;b. 确认罐中的缺氧环境。厌氧指标的颜色在缺氧条件下, 常氧环境中的浅蓝色变化为白白色。<a href="https://www.jove.com/files/ftp_upload/57725/57725fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57725/57725fig2.jpg"> 图2。用流式细胞仪验证建立缺氧/复氧 (h-/R) 模型的有效性.与正常组相比, h-/R 组心肌细胞凋亡率显著增高, 而通心络逆转这一变化 (p < 0.05 与正常; # p < 0.05 vs H/R; n = 3)11。<a href="https://www.jove.com/files/ftp_upload/57725/57725fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57725/57725fig3.jpg"> 图3。在 SSC 与 FSC 点图进行进一步分析时, 浇口细胞数量 (P1, 红色).使用侧向散射 (SSC) 和正散射 (FSC) 评估细胞种群的完整性。细胞碎片 (门外 P1, 黑色), 减少光散射, 被排除。<a href="https://www.jove.com/files/ftp_upload/57725/57725fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57725/57725fig4.jpg"> 图4。人心肌细胞线粒体膜电位的评价.与正常组相比, h-/R 组显示的红/绿比值显著降低, 而通心络扭转这种趋势 (红: 无荧光细胞;蓝色, 细胞与红色荧光;紫色, 细胞与绿色荧光。阴性控制, 空白, 无染色;阳性对照, 细胞 CCCP 治疗;*p < 0.05 与正常;#p < 0.05 vs/R;n = 3)11。<a href="https://www.jove.com/files/ftp_upload/57725/57725fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation at JoVE.com

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

在这里, 我们提出一个协议, 使用 JC-1 染料评估细胞线粒体膜电位后, 暴露在缺氧/复氧与或没有保护剂。

流式细胞仪测定缺氧/复氧后心肌细胞线粒体膜电位的实验研究

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

a-flow-cytometry-based-assay-for-measuring-mitochondrial-membrane

Nanyang Technological University

Research

JoVE Journal

Medicine

14.2K 次观看.  Chinese Academy of Medical Sciences and Peking Union Medical College. 在这里, 我们提出一个协议, 使用 JC-1 染料评估细胞线粒体膜电位后, 暴露在缺氧/复氧与或没有保护剂。

视频: A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

稳定同位素追踪和LC-MS分析以测量DHODH和SDH活性

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

作者聚焦：测量哺乳动物线粒体功能的氧非依赖型检测  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

测量哺乳动物线粒体功能的氧非依赖性测定

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

科研

生物化学

Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis.
The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture.
This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.

Saponin-permeabilized fiber preparation in conjunction with respirometric oxidative phosphorylation analysis provides integrative assessment of mitochondrial function. Mitochondrial respiration in physiological and pathological states can reflect various regulatory influences including mitochondrial interactions, morphology and biochemistry.

Respirometric皂甙通透心脏纤维的氧化磷酸化的评估

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, ...

生物学

<meta charset="utf8"></head><body>刷蜥蜴肝线粒体中的线粒体功能评估

Assessment of Mitochondrial Oxygen Consumption Using a Plate Reader-based Fluorescent Assay

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of metabolic pathways. Therefore, experimentally assessing mitochondrial functionality can provide insight into variations among different populations or disease states. Additionally, it is valuable to assess whether isolated mitochondria are healthy enough to proceed with experiments. One characteristic often used to compare mitochondrial function in different samples is the rate of oxygen consumption. Oxygen consumption and subsequent calculation of the respiratory control ratio in either intact cells or mitochondria isolated from tissue can serve all three purposes. Using mitochondria isolated from the livers of brush lizards in conjunction with a phosphorescent probe that is sensitive to the fluctuations in oxygen concentration of a solution, we measured oxygen consumption using a fluorescent plate reader. This method is not only quick and efficient but also can be conducted with a small amount of mitochondria and without the need for specialized equipment. The step-by-step protocol described here increases the accessibility of mitochondrial functional assessment to researchers.

<meta charset="utf8"></head><body>使用基于读板器的荧光测定法评估线粒体耗氧量

Assessment of oxygen consumption provides integral information about mitochondrial function. Using a phosphorescent probe with a fluorescent plate reader, accurate and reproducible data can be obtained easily without specialized equipment. This assay enables any lab to measure the oxygen consumption of isolated mitochondria and calculate respiratory control ratios.

使用基于读板器的荧光测定法评估线粒体耗氧量

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

细胞外通量分析仪分析原代培养小鼠新生心肌细胞的耗氧率

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an ...

医学

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

流式细胞仪测定缺氧/复氧后心肌细胞线粒体膜电位的实验研究

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小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

细胞外通量分析仪分析原代培养小鼠新生心肌细胞的耗氧率

外层膜蛋白的分离线粒体的流式细胞仪免疫检测