我们感谢劳瑞Destroismaisons和出色的技术支持莎拉Peyrard和Dr.Alexandre宝勒访问的流式细胞仪。我们也想感谢蒂莫西·米勒博士，他的贡献就从筹备去除髓鞘。这项工作是由卫生研究院（CIHR）神经肌肉研究合作伙伴，加拿大创新基金会，加拿大ALS协会，基金会弗里克ALS研究，密友基金会和全宗德拉RECHERCHE桑特连接魁北克（CVV）加拿大研究院的支持。无论是CVV和NA是研究学者的全宗德拉RECHERCHE桑特连接魁北克与CIHR新的调查。 SP被蒂姆·诺埃尔助学金来自加拿大的ALS协会的支持。

在这项研究中使用的动物严格按照协议（N08001CVsr）批准的中心德RECHERCHE杜中心医院DE L&#39;蒙特利尔大学（CRCHUM）机构委员会动物保护的它遵循概述了加拿大国家标准进行治疗理事会动物护理（CCAC）。 准备执行该协议（ 表1）所需要的所有试剂。关于设备，耗材及供应商的所有其他细节可以在材料清单上找到。 <img alt="表1" fo:content-width="5in" src="/files/ftp_upload/51887/51887table1.jpg" width="500" /> 表1缓冲区组成。 1，收集大鼠脊髓<ol><li>深深地麻醉大鼠（只SD）与4％异氟烷。在捏个的验证anaesthetization因缺乏反射ê前爪。通过断头台安乐死的大鼠断头。安乐死该方法优于其他，这可能会歪曲脊髓。 </li><li>切开背部皮肤，以暴露脊柱。切骨剪略高于骨盆脊柱。可视化的脊柱开幕。 </li><li>将10毫升注射器，用200微升枪头（通过在火焰稍微融化附后），充满磷酸盐缓冲液（PBS），到脊柱。 </li><li>通过施加的压力的培养基中量到柱塞冲洗出脊髓。 </li><li>如果任何血液存在于脊髓，在进行下一步骤之前，漂洗用PBS。 注:此方法也被证实为脑和肝。如果包括这些组织中，收集半从安乐死的大鼠的脑和一片肝相等的权重至脊髓。所有其他步骤保持一致。 </li></ol> 2，隔离脊髓麻省理工学院ochondria（改编自范德维尔德等。4） <ol><li>收集整个完整的脊髓和地点与5卷（〜3.25毫升）匀浆缓冲液（HB）5毫升玻璃匀浆器。对于分离线粒体的最佳恢复，冰或冷室执行所有步骤。用手均匀的组织，直到没有大块的组织仍然存在，大约8招。置于匀浆中的两（2毫升），三（1.7毫升）的微量离心管中。离心1300 XG在4℃的台式离心10分钟。 </li><li>回收上清液并置于5ml的超离心管。加入750微升（约0.5倍体积）HB到含有颗粒状的离心管中，轻轻地重悬沉淀。重复离心和重悬步骤两次以上。池中的所有上清液（S1A和S1B）到同一个5毫升超速上面管。此步骤用于去除小碎片。 </li><li>汇集离心机使用S1配备了吊桶式腐超速离心机或离心在17000×g离心15分钟，在4℃。 </li><li>保留上清液（S2），用于进一步的处理，如果胞浆部分感兴趣（例如，用于Western印迹分析）。重悬沉淀（P2）的粗线粒体级分，在4毫升HB +的50mM的KCl。离心17000×g离心4℃15分钟的水平转头。弃上清，轻轻悬浮在800微升乙肝颗粒（P3）。 注:线粒体是用HB +的50mM的KCl，以除去任何非特异性线粒体相关的污染物。 </li><li>在新的5毫升超速离心管，加入重悬沉淀（P3），正好800微升。 </li><li>向该试管中加入200微升碘克沙醇（密度梯度介质），从而产生12％的碘克沙醇的终浓度。轻轻的，但通过彻底吸取了P1000的混合管的内容。此外碘克沙醇的第一，然后再悬浮沉淀（P3）可以是优选的，以便充分混合。离心机的ULTracentrifuge在4℃下配备吊桶式转子中以17000×g离心15分钟。 </li><li>注:肝不包含髓鞘，因此，如果仅在肝脏正在处理这一步骤不是必需的。但是，如果肝并发中枢神经系统线粒体进行处理，建议同等对待所有的组织。 </li><li>吸髓磷脂的层在该管的顶部，小心地取出并丢弃上清液。球团可能松动。悬浮在4ml HB沉淀。再次离心在17000 XG在4℃下10分钟。弃上清，悬浮在4毫升HB沉淀。重复离心，去除上清。 </li><li>悬浮最终沉淀（P7）在100-200微升HB和转移到1.7 ml离心管。此示例包含分离线粒体。 </li><li>继续进行蛋白定量。稀释样品并在2％十二烷基硫酸钠（SDS）的标准曲线，以确保线粒体杜里充分溶吴蛋白定量。 </li></ol> 3，免疫标记分离线粒体的流式细胞仪<ol><li>对于每一个染色组合进行测试，吸管25微克分离线粒体到1.7 ml离心管。包括在每个实验未染色的样品;并为每个抗体，一定要包括相应的​​同型对照样本。 </li><li>离心机在17000×g离心2分钟，在4℃下，在台式离心。 </li><li>除去上清，悬浮分离的线粒体在50μl线粒体缓冲液（M缓冲液）于4℃（封闭步骤）补充有10％无脂肪酸BSA的15分钟。 注:在标签，在冰箱中4℃进行孵化。 </li><li>第一抗体（兔抗-Mfn2的，每毫升含20微克）加入到管中，并温育30分钟，在4℃下。 注:用滴定法确定每个抗体的最佳浓度经验。由于变异浓度和/或对urity，不同批次来自同一制造商的同一抗体可能导致不同的结果;因此滴定需要为每个新的大量抗体。 </li><li>在4℃离心17000×g离心2分钟:洗出未结合的抗体。除去上清液，并轻轻悬浮于200μl缓冲液M的颗粒。离心机在5185 XG在4℃下2分钟。除去上清液，重悬在50微升M缓冲沉淀。 </li><li>第二抗体（驴抗兔IgG藻红蛋白（PE），0.5微克每毫升）添加至试管孵育样品30分钟，在4℃，避光。 </li><li>在4℃离心17000×g离心2分钟:洗出未结合的二级抗体。弃上清，重悬在200微升M缓冲沉淀。离心机在5185 XG在4℃下2分钟。除去上清液，重悬在500μlM缓冲沉淀。 </li><li>为确保活动其实都是线粒体，染色分离线粒体用线粒体特异性荧光染料15分钟，在室温下，避光。如果其他功能参数（线粒体跨膜电位或过氧化产物）染色的需要，继续执行步骤4。如果没有，请继续收购步骤5。 注意:验证该第二抗体的发射光谱是与该功能性染料的相容是很重要的。例如，如果验证线粒体纯度用市售染料和类似FITC和跨膜电位与四甲甲酯（TMRM）的光谱特性，一个可行的二级抗体将别藻蓝蛋白（APC:实施例650纳米/ EM 660纳米）。添加补偿控制， 即免疫标记或染色用一个荧光基团，在适用的样本。 </li><li>转移到适合装载流式细胞仪的管子。 （为方便样本量小，微量滴定管放置在3毫升流式细胞仪管。）保持样品在冰上，并立即着手流式细胞仪的采集。 </li></ol> 4，含量测定线粒体跨膜电位与线粒体超氧化物生成流式细胞仪<ol><li>验证分离的线粒体通过用100nM TMRM（实施例548纳米/ EM 574纳米）5，染色在步骤3.8，为15分钟，在室温下，避光有一个完整的跨膜电位。对样品和种群，使用TMRM（1〜30纳米）的下部/非淬火浓度之间的跨膜电位的比较，可以更适当6。 <ol><li>至于TMRM染色，染色分离线粒体与100纳米TMRM在100微米羰基氰氯苯肼（CCCP）的存在，线粒体解耦联，将线粒体去极化控制。 CCCP的去极化的线粒体所需的浓度可以小于，如果使用较低的染料的浓度。 </li></ol></li><li>验证分离线粒体产生线粒体超氧由圣用合适的线粒体超氧化物指示器7癌宁，也是在步骤3.8，为15分钟，在室温，避光。 <ol><li>作为线粒体超氧化物的产生，色斑分离线粒体用染料在10μM抗霉素A，呼吸链，这将增加线粒体超氧化物生成的复合物III抑制剂的存在下的控制。 </li></ol></li></ol> 5，采集免疫标记与分析分离线粒体流式细胞仪<ol><li>仪器设置:从线性电压切换登录方式，以利分离线粒体，并设置电压的分析（FSC:450; SSC:250）。确保事件被收集在FSC-A（面积）模式以及FSC-W（宽度）和FSC-H（高度），以便能够排除双峰（ 即，两个事件，通过检测器传递的同时）在分析后的数据集合。事件的张数设定为被收集到100,000。获得补偿控制（如适用）。 </li><li>数据采集​​:数据采集前，避免涡旋样本。代替通过轻轻敲击管混匀。最初收集在低压活动，浇注过程中。门上的总人口。调整直方图的电压相应地，通常是未染色的样品的峰值将对应于第二个十年（10 2）。一旦门被建立，并且样品正在处理中，压力可切换到高。 </li><li>分析:通过绘制FSC-W与FSC的可视化双峰。确定汗衫及双峰。门上的汗衫。通过门控上都与线粒体选择性染料染色阳性事件选择线粒体人口。 </li><li>确定由同型对照背景标记。使用同种型对照样品，测定线粒体人口标记阳性Mfn2的抗体的百分比。 </li></ol>

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作者什么都没有透露。

这是越来越明显，线粒体是关键球员在正常生理和疾病。虽然免疫可以确定哪些蛋白质在线粒体或在线粒体表面发现了在一定的条件下，对​​整个人口的平均这个方法报告。此方法不能产生约线粒体亚群或亚群的相对丰度信息。虽然先前已假设所有线粒体创建相等，则该字段被越来越多地认识到在小区内的线粒体在形态和/或函数10方面的广泛变化。 荧光显微镜方法...

线粒体是经过多轮的裂变和聚变，被运到高能源需求的网站，并迅速生理刺激反应1高动态性的细胞器。因为人们越来越认识到不同组织内的线粒体，甚至不同的细胞区室，具有不同的功能配置文件，需要新的方法来识别这些不同的线粒体亚群。 显微术提供了一种方法，通过个别线粒体可以可视化和蛋白质在或在线粒体可以通过免疫2来确定是否存在。然而，用这种方法定量分析是劳动密集和更适合用永生化或原代细胞系的实验。来源于组织的个体的线粒体的研究是显著更加困难和最方法不允许以易于识别线粒体亚群与evalu并发通报BULLETIN线粒体功能3。 为了解决这一难题，一种新颖的方法来immunolabel线粒体分离从啮齿动物组织中，并随后分析通过流式细胞术已经被开发出来。这允许快速检测并定位于线粒体外膜，其相比于分析由显微镜蛋白质的定量，少得多的劳动密集的，并允许以十万计的线粒体中的单个样品的分析。此测定法可用于监测被认为是组成性存在于线粒体的线粒体外膜蛋白质的命运和相对量，蛋白质的募集到线粒体表面，或错误定位在病理条件下，线粒体蛋白的检测。此外，以往的荧光指示剂染料的掺入允许的线粒体功能的某些方面，在不同的线粒体同时评价亚群。

<table><tbody><tr><td>Rats</td><td>Charles River</td><td>Strain code 400</td><td>Adult (9 weeks to 18 weeks) male or female rats can be used for the isolation protocol. Weight of rats is dependent on gender and age (males between 300 to 500 g and females between 200 to 350 g) are typically used.</td></tr><tr><td>Dounce homogenizer</td><td>Kontes Glass Co.&amp;nbsp;</td><td>885450-0022</td><td>Duall 22</td></tr><tr><td>Microcentrifuge</td><td>Thermo Scientific</td><td></td><td>Sorvall Legend Micro 17 R</td></tr><tr><td>Ultra-Clear Ultracentrifuge tubes</td><td>Beckman Coulter</td><td>344057</td><td>Transparent, thin walled&amp;nbsp;</td></tr><tr><td>Sorvall Ultracentrifuge</td><td>Thermo Scientific</td><td></td><td>Sorvall WX UltraSeries</td></tr><tr><td>AH-650 rotor and buckets&amp;nbsp;&amp;nbsp;</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Opti-prep</td><td>Axis-Shield</td><td>1114542</td><td>Iodixanol, density gradient medium</td></tr><tr><td>Fatty acid free Bovine Serum Albumin</td><td>Sigma</td><td>A8806</td><td>Must be fatty acid free for mitochondria</td></tr><tr><td>Sodium succinate dibasic hexahydrate&amp;nbsp;&amp;nbsp;</td><td>Sigma</td><td>S9637</td><td></td></tr><tr><td>Rabbit anti-Mitofusin2 antibody</td><td>Sigma</td><td>M6319</td><td></td></tr><tr><td>Rabbit IgG</td><td>Jackson Immuno Research&amp;nbsp;</td><td>011-000-003</td><td></td></tr><tr><td>Anti-Rabbit IgG PE</td><td>eBioscience</td><td>12-4739-81</td><td></td></tr><tr><td>Micro titer tube</td><td>Bio-Rad</td><td>223-9391</td><td>For sample acquisition by flow cytometry&amp;nbsp;</td></tr><tr><td>MitoTrackerGreen (MTG)</td><td>Invitrogen</td><td>M7514</td><td>100 nM: Ex 490 nm/Em 516 nm</td></tr><tr><td>TMRM</td><td>Invitrogen</td><td>T668</td><td>100 nM: Ex 548 nm/Em 574 nm</td></tr><tr><td>CCCP</td><td>Sigma</td><td>C2759</td><td></td></tr><tr><td>MitoSOX Red</td><td>Invitrogen</td><td>M36008</td><td>5 &amp;micro;M: Ex 540 nm/Em 600 nm</td></tr><tr><td>Antimycin A</td><td>Sigma</td><td>A8874</td><td></td></tr><tr><td>LSR II flow cytometer</td><td>BD</td><td></td><td></td></tr><tr><td>BD FACSDiva Software</td><td>BD</td><td></td><td></td></tr><tr><td>FlowJo</td><td>TreeStar Inc.&amp;nbsp;</td><td></td><td>Software used for analysis</td></tr><tr><td>BCA protein assay kit&amp;nbsp;&amp;nbsp;</td><td>Pierce/Thermo Scientific</td><td>23225</td><td>Bradford assay is not recomended as it is not compatible with high concentrations of SDS</td></tr></tbody></table>

immunodetection of outer membrane proteins by flow cytometry of isolated mitochondria

方法来检测和监测线粒体外膜蛋白成分在动物组织是研究线粒体生理和病理生理至关重要。该协议描述了线粒体啮齿动物组织中分离出免疫标记是通过流式细胞仪检测的技术。线粒体是从啮齿动物的脊髓中分离并进行快速浓缩步骤，以除去髓磷脂，来自神经组织制备的线粒体级分中的主要污染物。分离的线粒体，然后标记所选择的抗体和荧光标记的二抗。分析流量通过与线粒体特异性染料，其次是检测和免疫标记蛋白定量染色流式细胞仪验证线粒体制剂的相对纯度。该技术具有快速，量化和高吞吐量，允许成千上万的每个样本的线粒体的分析。它可适用于评估新颖p在正常生理条件下，在线粒体表面roteins以及病理过程中，可能会成为错误定位到该细胞器的蛋白质。重要的是，这种方法可以被耦合到荧光指示剂染料对线粒体亚群的某些活动的报告，并为从中枢神经系统（脑和脊髓）以及肝线粒体可行的。

从大鼠脊髓源性线粒体可以免疫标记与靶向Mitofusin2（Mfn2的），牵连线粒体8的外膜的融合蛋白的抗体。以下隔离和标签带Mfn2的特异性抗体和荧光标记的二抗，线粒体通过流式细胞术（ 图1）进行处理。以下的数据采集，样品是使用流式细胞分析软件，由第一可视化上的点图（ 图2A）的所有收集的事件进行分析。当事件被绘制在FSC，宽度（W）相对于面积（A）（ <str...

Watch this Scientific Journal Video about Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria at JoVE.com

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

这里描述的是这样一种方法来检测并通过免疫标记线粒体分离从啮齿动物的组织和通过流式细胞仪分析的量化线粒体外膜蛋白质。该方法可以被扩展以评估线粒体亚群的功能方面。

外层膜蛋白的分离线粒体的流式细胞仪免疫检测

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

immunodetection-outer-membrane-proteins-flow-cytometry-isolated

Research

JoVE Journal

Biology

15.5K 次观看.  Université de Montréal, CRCHUM. 这里描述的是这样一种方法来检测并通过免疫标记线粒体分离从啮齿动物的组织和通过流式细胞仪分析的量化线粒体外膜蛋白质。该方法可以被扩展以评估线粒体亚群的功能方面。 

视频: Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

拟南芥线粒体的分离和呼吸测量

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

科研

生物化学

分离线粒体接触位点

An Improved Method to Isolate Mitochondrial Contact Sites

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the synthesis of iron-sulfur clusters, lipids, or proteins, Ca2+ buffering, and the induction of apoptosis. Likewise, mitochondrial dysfunction results in severe human diseases such as cancer, diabetes, and neurodegeneration. In order to perform these functions, mitochondria have to communicate with the rest of the cell across their envelope, which consists of two membranes. Therefore, these two membranes have to interact constantly. Proteinaceous contact sites between the mitochondrial inner and outer membranes are essential in this respect. So far, several contact sites have been identified. In the method described here, Saccharomyces cerevisiae mitochondria are used to isolate contact sites and, thus, identify candidates that qualify for contact site proteins. We used this method to identify the mitochondrial contact site and cristae organizing system (MICOS) complex, one of the major contact site-forming complexes in the mitochondrial inner membrane, which is conserved from yeast to humans. Recently, we further improved this method to identify a novel contact site consisting of Cqd1 and the Por1-Om14 complex.

作者聚焦:揭开线粒体接触位点和架构见解 

Mitochondrial contact sites are protein complexes that interact with mitochondrial inner and outer membrane proteins. These sites are essential for the communication between the mitochondrial membranes and, thus, between the cytosol and the mitochondrial matrix. Here, we describe a method to identify candidates qualifying for this specific class of proteins.

一种分离线粒体接触位点的改进方法

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

免疫与感染

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

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment elevated myocardial infarction. However, reperfusion per se can result in further cardiomyocyte death, a phenomenon known as reperfusion injury. The opening of the mitochondrial permeability transition pore (mPTP), with the decrease of the mitochondrial membrane potential (MMP), or mitochondrial depolarization, is universally recognized as the final step of reperfusion injury and is responsible for mitochondrial and cardiomyocyte death. JC-1 is a lipophilic cationic dye that accumulates in mitochondria depending on the value of MMP. The higher the MMP is, the more JC-1 accumulates in the mitochondria. The increasing amounts of JC-1 in mitochondria can be reflected by a fluorescence emission shift from green (~530 nm) to red (~590 nm). Therefore, the reduction of the red/green fluorescence intensity ratio can indicate the depolarization of mitochondria. Here, we take advantage of JC-1 to measure MMP, or the opening of mPTP in human cardiac myocytes after hypoxia/reoxygenation, detected by flow cytometry.

Here, we present a protocol that uses JC-1 dye to assess the mitochondrial membrane potential of cells after being exposed to hypoxia/reoxygenation with or without a protective agent.

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 ...

医学

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

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.

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.

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

生物学

Application of Flow Cytometric Analysis for Measuring Multiple Mitochondrial Parameters in 3D Brain Organoids

Mitochondrial dysfunction is a common primary or secondary contributor to many types of neurodegeneration, and changes in mitochondrial mass, mitochondrial respiratory chain (MRC) complexes, and mitochondrial DNA (mtDNA) copy number often feature in these processes. Human brain organoids derived from human induced pluripotent stem cells (iPSCs) recapitulate the brain&#39;s three-dimensional (3D) cytoarchitectural arrangement and offer the possibility to study disease mechanisms and screen new therapeutics in a complex human system. Here, we report a unique flow cytometry-based approach to measure multiple mitochondrial parameters in iPSC-derived cortical organoids. This report details a protocol for generating cortical brain organoids from iPSCs, single-cell dissociation of generated organoids, fixation, staining, and subsequent flow cytometric analysis to assess multiple mitochondrial parameters. Double staining with antibodies against the MRC complex subunit NADH: Ubiquinone Oxidoreductase Subunit B10 (NDUFB10) or mitochondrial transcription factor A (TFAM) together with voltage-dependent anion-selective channel 1 (VDAC 1) permits assessment of the amount of these proteins per mitochondrion. Since the quantity of TFAM corresponds to the amount of mtDNA, it provides an indirect estimation of the number of mtDNA copies per mitochondrial content. This entire procedure can be completed within a span of 2-3 h. Crucially, it allows for the concurrent quantification of multiple mitochondrial parameters, including both total and specific levels relative to the mitochondrial mass.

<meta charset="utf8"></head><body>流式细胞术分析在测量 3D 脑类器官中多个线粒体参数的应用

This protocol reports a unique method of using a streaming cytometer and multiple antibodies for simultaneous assessment of multiple mitochondrial functional parameters, including changes in mitochondrial volume, amounts of the mitochondrial respiratory chain (MRC) complex subunits, and mitochondrial DNA (mtDNA) replication.

流式细胞术分析在测量 3D 脑类器官中多个线粒体参数的应用

Mitochondrial dysfunction is a common primary or secondary contributor to many types of neurodegeneration, and changes in mitochondrial mass, ...

神经科学

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

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.

外层膜蛋白的分离线粒体的流式细胞仪免疫检测

摘要

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