Name: using isolated mitochondria from minimal quantities of mouse skeletal muscle for high throughput microplate respiratory measurements
Uploaded: 2015-10-30T14:00:00.000+00:00
Duration: 10 min 39 s
Description: Watch this Scientific Journal Video about Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements at JoVE.com

The Fralin Life Science Research Institute and The Metabolic Phenotyping Core at Virginia Tech supported this work.

注:此协议开始后的一个已分离出红骨骼肌线粒体。 100毫克红色骨骼肌13 -线粒体如先前从〜75描述分离。在5 - 10微克/微升线粒体蛋白会从这一数额的组织重新悬浮最后的线粒体沉淀于≤50微升隔离缓冲线粒体（IBM）2（见Frisard 等 13）来实现。 1.安装<ol><li>制备工作储备溶液（表1）和线粒体测定溶液（MAS）混合（ 表2）用于随后的测定。 注意:大多数的准备的测定的可预先完成，因为大多数股票溶液和MAS可以被存储。 </li><li>在37℃实验中一个非 CO 2培养箱前水合在1ml溶液校准一个呼吸计测定盒过夜。 </li><li>的实验中，吨的天阿克出冷冻股票（基材和线粒体调制器，MAS组合，MAS W / O BSA， 表1），解冻37℃水浴或孵化器。 </li><li>准备所有底物溶液（表3）和注射剂（表4-5）和根据需要（pH7.4）中调节pH。后调节pH，储存在冰上。 </li><li>程序的多井耗氧量测定器，以适当的组合，等待，测量参数，首先进入分析软件，其次是"标准"模式（ 见表 6）和标签背景和组/条件井。 <ol><li>进入"客人"论坛，并点击"分析向导"。曾经在"试验向导"，单击"协议"，以更改组合，等待，测量参数。标签下方的"回到后台井。更正"选项卡，一定要突出"做背景校正"。 </li><li>标签CONDitions首先点击"组和标签"选项卡上，其次是"组信息"选项卡。当这些参数被输入后，点击"结束"，其次是"保存模板"。 </li></ol></li></ol> 2.试验运行<ol><li>加载注射液成分析来看墨盒，首先进入分析软件，然后输入"标准"模式开始校准。进入"访客"论坛。打开"XF"的驱动下，保存在步骤1.5的模板，"模板"选项卡下。一旦打开，请单击"开始"，开始校准。 注:该试剂盒盒式运行必须输入到机器的缺角的左下角。这大约需要30分钟。小心注入解决方案集成到正确的端口。 表 4-5注射顺序列出加载。 </li><li>轻轻地，彻底混合和线粒体河口股票通过搅拌溶液与200μl的移液管尖，随后轻轻捣碎股票用200μl的移液管尖端有其孔口通过切割〜3毫米用剪刀尖端的端部变宽。 </li><li>执行双金鸡宁酸（BCA）测定法来确定库存线粒体的蛋白质浓度。 </li><li>使用来自步骤2.3获得的库存浓度，重悬10微克线粒体蛋白/ 200微升琥珀酸/鱼藤酮底物混合物（表3）。重悬14线粒体蛋白的微克/ 200微升剩余基板混合物的（必须为每个衬底混完成）（见表3）。置于冰上所有的线粒体蛋白/底物混合。 注意:每孔线粒体蛋白的每个测定的最佳量如前面所述9确定。经测定，丙酮酸/苹果酸盐，棕榈酰肉碱/苹果酸盐，谷氨酸/苹果酸盐的ð电子流测定利用3.5微克线粒体蛋白，而琥珀酸盐/鱼藤酮测定法利用2.5微克线粒体蛋白的每孔。因此，研究人员需要重悬足够线粒体蛋白为4个重复在此步骤，因为无论是2.5微克或每50μl3.5微克每孔加载（参见步骤2.6）。 </li><li>轻轻地，但彻底搅拌溶液用200微升的吸头，接着轻轻捣碎的股票有200微升枪头，这带来了〜3毫米其末端的混合线粒体/底物溶液切断。 </li><li>放置一个细胞培养板在冰上，一式三份，负载50μl/孔的各线粒体/基材的混合物。在基于微孔板透气性检测的开发者推荐留出至少两个空白（无线粒体）井，最好在细胞培养板两侧。 </li><li>旋细胞培养板在2000克20分钟，在4℃。虽然板为s穿针，预热底物溶液在37℃水浴。 </li><li>自旋完成后，小心地加载450微升在其相应孔中的顶端各底物溶液（即，加载丙酮酸/苹果酸盐衬底具有线粒体最初悬浮于该衬底溶液的孔中）。一定要在室温加载板。注意:空白孔应装有500微升底物。指定用于电子流动化验空白孔应加载与电子流基底（丙酮酸/苹果酸+ FCCP），而耦合测定空白孔中可以装入任何耦合测定衬底。 注意:如果在执行耦合和电流测定在同一板上，研究者必须重新分配背景孔于各自试验测定运行完成经由"仪器"标签使用"XF读者"平台之后。一旦中了"仪器"的设置，点击"管理"标签，其次是"背景校正"。点击"结束仪器设置模式"完成时。这是因为抗坏血酸/ TMPD的组合可以消耗的 O 2，因此单独的背景校正，需要对每个检测类型。 </li><li>后450微升底物加入到每个孔中，查看线粒体在阱的层，以确保线粒体均匀地分散在单个单层（20X放大率〔见罗杰斯9， 图4]）。没有出现有均匀分布的单层井可以除去事后 。 </li><li>检查线粒体坚持后，将微孔板放入透气性测定仪，并开始通过点击"OK"运行试验。 注意:从步骤2.1标定必须完成之前运行将开始。一旦研究人员点击"确定"后，机器会弹出用于校准的工具盘。 <ol> &lt;利&gt;取出实用程序板和放置包含该托盘上的线粒体的细胞培养板。蓝色槽口上的细胞培养板应放置在托盘的左下角。点击"继续"开始试验运行。 </li></ol></li><li>在运行完成后，通过在屏幕上按下"弹出"弹出细胞培养板和墨盒。 <ol><li>一旦板被弹出，捞出弃去细胞培养板和墨盒，在屏幕上点击"继续"。该运行将被自动保存为"数据"文件中的.xls文件。 </li></ol></li><li>打开此文件并通过点击向下箭头下的改变，从"02"到"OCR"显示"Y1:"标记。 <ol><li>接下来的变化"中间点"到"点至点价格"下的"速率数据显示为:"选项。点击"确定"以应用这些更改。 </li></ol&gt; </li><li>点击屏幕上的类似条件井组一起左下角的"井组模式"选项卡。最后，点击"样本均值/标准差"选项卡向屏幕的中间，向左。另外，这些命令可以设置前的试验开始于"试验向导"的设置。 注意:每个条件将具有平均值±显示每个速率和每个条件标准偏差。还有每个状态（状态2 [联轴器] / State3u [电子流]呼吸后面四次注射）采取率测量。使用的状态2速率的第二测量的平均值，确定国家的4o在最低点寡注射后，使用最大点为状态3和状态3U ADP和FCCP注射，分别后，并使用最小点为抗霉素阿诱导呼吸的耦合检测。使用最大点丙酮酸/苹果酸诱导状态3U呼吸，用最小点为鱼藤酮诱导的呼吸，使用最大点琥珀诱导状态3U呼吸，并使用最小点为抗霉素A诱导呼吸的电子流测定法。所有实验都进行3次并示出的数据是一代表跟踪的结果。 </li></ol>

<ol>
	<li>Brand, M., Nicholls, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+mitochondrial+dysfunction+in+cells.">Assessing mitochondrial dysfunction in cells.</a> Biochem J. 435, 297-312 (2011).</li><li>Wang, H., Hiatt, W. R., Barstow, T. J., Brass, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+between+muscle+mitochondrial+DNA+content,+mitochondrial+enzyme+activity+and+oxidative+capacity+in+man:+alterations+with+disease.">Relationships between muscle mitochondrial DNA content, mitochondrial enzyme activity and oxidative capacity in man: alterations with disease.</a> European Journal of Applied Physiology and Occupational Physiology. 80, 22-27 (1999).</li><li>Lauri, A., Pompilio, G., Capogrossi, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+genome+in+aging+and+senescence.">The mitochondrial genome in aging and senescence.</a> Ageing Research Reviews. 18C, 1-15 (2014).</li><li>Nunnari, J., Suomalainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+in+sickness+and+in+health.">Mitochondria: in sickness and in health.</a> Cell. 148, 1145-1159 (2012).</li><li>Dube, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+acute+lipid+overload+on+skeletal+muscle+insulin+resistance,+metabolic+flexibility,+and+mitochondrial+performance.">Effects of acute lipid overload on skeletal muscle insulin resistance, metabolic flexibility, and mitochondrial performance.</a> Endocrinology and Metabolism. 307, E1117-E1124 (2014).</li><li>Disease Control and Prevention Services. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=National+diabetes+statistics+report:+estimates+of+diabetes+and+its+burden+in+the+United+States.">National diabetes statistics report: estimates of diabetes and its burden in the United States.</a> , (2014).</li><li>Guarino, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+determining+oxygen+consumption+rates+of+static+cultures+from+microplate+measurements+of+pericellular+dissolved+oxygen+concentration.">Method for determining oxygen consumption rates of static cultures from microplate measurements of pericellular dissolved oxygen concentration.</a> Biotechnology and Bioengineering. 86, 775-787 (2004).</li><li>Will, Y., Hynes, J., Ogurtsov, V. I., Papkovsky, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mitochondrial+function+using+phosphorescent+oxygen-sensitive+probes.">Analysis of mitochondrial function using phosphorescent oxygen-sensitive probes.</a> Nature Protocols. 1, 2563-2572 (2007).</li><li>Rogers, G. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+microplate+respiratory+measurements+using+minimal+quantities+of+isolated+mitochondria.">High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria.</a> PloS One. 6, e21746 (2011).</li><li>Wu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparameter+metabolic+analysis+reveals+a+close+link+between+attenuated+mitochondrial+bioenergetic+function+and+enhanced+glycolysis+dependency+in+human+tumor+cells.">Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells.</a> American Journal of Physiology-Cell Physiology. 292, C125-C136 (2007).</li><li>Fernandez-Vizarra, E., Lopez-Perez, M. J., Enriquez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+biogenetically+competent+mitochondria+from+mammalian+tissues+and+cultured+cells.">Isolation of biogenetically competent mitochondria from mammalian tissues and cultured cells.</a> Methods (San Diego, Calif.). 26, 292-297 (2002).</li><li>Frezza, C., Cipolat, S., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+isolation:+functional+mitochondria+from+mouse+liver,+muscle+and+cultured+fibroblasts.">Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts.</a> Nature Protocols. 2, 287-295 (2007).</li><li>Frisard, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+levels+of+lipopolysaccharide+modulate+mitochondrial+oxygen+consumption+in+skeletal+muscle.">Low levels of lipopolysaccharide modulate mitochondrial oxygen consumption in skeletal muscle.</a> Metabolism. 64, 416-427 (2015).</li><li>Chance, B., Williams, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+respiratory+chain+and+oxidative+phosphorylation.">The respiratory chain and oxidative phosphorylation.</a> Advances in Enzymology and Related Subjects of Biochemistry. 17, 65-134 (1956).</li><li>Gnaiger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+pathways+and+respiratory+control.">Mitochondrial pathways and respiratory control.</a> , (2007).</li></ol>

乔治·W·罗杰斯是海马生物科学的员工是生产的这些分析是开发的工具。

在这篇文章中介绍的方法提供一步一步的指示，使用线粒体75隔绝基于微孔板检测呼吸速率集合的性能 - 100毫克的小鼠骨骼肌。这些测定法可以以高精度来执行如由一式三份孔之间的紧密标准偏差。重要的是，这些透气性测定法允许精确定位，其中异常和/或改编可在通常使用的动物模型可发生在ETC，TCA循环，β氧化途径，衬底转运等。 它突出使用在本协议中使用的各种燃料和抑制剂的基本原理是很重要的。丙酮酸/苹果酸和谷氨酸/苹果酸盐呼吸计测定允许复合物I介导的呼吸的评估，以及它们各自的转运体的评估，以及在谷氨酸的情况下，该脱氨酶15。可替代地，组合琥珀酸/鱼藤酮使线粒体呼吸通量通过ETC的复合物II的评估，因为鱼藤酮抑制复杂我和琥珀酸通过黄素腺嘌呤二核苷酸的还原提供电子向复合物II（FADH 2）15。这些实验提供基板的具体信息以耦合效率和最大呼吸。电子流测定法是在底物和抑制剂的组合允许多种复合物的线粒体呼吸焊剂9在评估是唯一的。丙酮酸/苹果酸+ FCCP的初始衬底混允许最大呼吸络合我驱动的评价，而鱼藤酮的注射随后琥珀酸允许通过复合物II从动评估最大呼吸。注射抗霉素A的，复合物III抑制剂，接着注射抗坏血酸/ TMPD允许为呼吸自抗坏血酸由复合物IV驱动的评价/ TMPD是一个电子供体细胞色素C /复合物IV。虽然获得的耦合效率没有信息，该方法适合于那些妨碍独立运行的多个基板非常小的样本量。最后，使用棕榈酰肉碱/苹果酸盐的允许β氧化和由于来自的棕榈酸（β氧化）的氧化所产生的还原当量的ETC之间的协调的评估送入ETC通过电子转移黄素蛋白15。应当指出的是，耦合和电流测定法也可用于在串联以确定改变线粒体功能，由于一些干预（药物治疗，基因操作）。 高精度实现对这些测定主要是由于线粒体的充分混合，无论是前蛋白测定，或与基板的解决方案。沿着这些线路，一旦线粒体是resuspendend在衬底搜索解决方案纳秒，关键是混合此溶液彻底之前按照步骤2.2中所述带的加宽孔枪头加载细胞培养板。失败来混合线粒体充分将导致在测定内大的变化。此外，使用一个窄孔移液管尖端将产生的剪切力，同时混合的线粒体并增加损伤线粒体膜和细胞色素C的释放的粘附步骤（2.7）也是在这个协议中的一个关键步骤的潜力。失败旋转加载的细胞培养板长足够快/将导致线粒体的不完全粘附到孔，因此导致油井和测量之间增加变性。 所描述的方案进行了优化，包括:装载线粒体蛋白质的最佳量每孔，使用正确的浓度/制备方法，使股市和基体的解决方案，改变了试验运行，确保线粒体状态解放军teaus，和线粒体库存和线粒体/衬底混的合适的混合。在此之前，优化的努力，作者在试验运行中遇到的陷阱。下面讨论的故障排除方法/是优化这个协议有帮助修改。对于最佳的装载，装载太少线粒体不会引起一个强有力的反应，同时加载了太多的线粒体会耗尽微容器内的氧气，导致不准确的测量。 Rogers等9条规定确定每口井的线粒体最佳负载量为基于微孔板透气性检测的例子。更多的时候，太多的线粒体是每个加载以及由国家证明2率超过100-200皮摩尔/分钟/孔，状态3速率&gt; 1500皮摩尔/分钟/孔。如果发生过载，进行一个实验，用不同浓度的线粒体蛋白（例如，在1 - 10微克），以动态R内部引起光学字符识别安格耗氧量测量机。准备和使用基材和个股的正确浓度是非常重要的。始终使用的基板/注射的酸形式和调整用氢氧化钾或盐酸的pH值;钠缓冲液/解决方案不推荐。此外，重悬基质/股在DMSO或100％的乙醇，将导致测量失败或错误。一定要使用95％的乙醇注意的地方。是很常见的棕榈酰肉碱以沉淀出95％的乙醇的解冻冷冻的储存后，从而导致大的可变性。一定要热身棕榈酰肉碱的股票和漩涡以及之前使用。此外，高度可变的丙酮酸/苹果酸盐化验结果，可能是由于丙酮酸股票之中&gt; 2周大。一定要重拍丙酮酸冷冻等份，每2周。该法的运行被修改为2分钟测量，以确保线粒体状态高原。如果观察ADP耗尽的需要，研究人员可该"协议"选项卡下延长​​测量时间下的"试验向导"的论坛。最后，当线粒体库存和线粒体加底物溶液不充分均质化在装载前发生大的变化。如果井间变异是试验运行后高，一定要在底物液充分搅拌下一个实验前。切勿涡线粒体/基板解决方案，而搅拌，捣烂，与加宽孔的枪头轻轻地磨碎。 有此技术是值得注意的一些限制。首先，孔中的用于这些分析的细胞培养板的数目是相对低的（即，24个孔和指定为空白孔至少2）。如果期望在一个板上执行所有5这些测定的，研究人员仅能够检查从一只小鼠的响应的时间。但是，应该指出的是，96孔仪器可用于解释第1条- [R吞吐量。其次，有先天的优势和劣势评估线粒体功能障碍的分离线粒体相比，在完整细胞1。一些弱点包括具有较少生理学相关性相比完整细胞和从隔离过程诱导工件。最后，该方法的成功是取决于线粒体隔离工艺的质量。 虽然这些测定法已被已显影在不同的系统或在其他动物模型中已被证实，在此提出的方法是首先在一个多井氧消耗测量机使用小鼠合成所有上述测定法的最佳利用骨骼肌。重要的是，所有5这些测定可以与线粒体从〜75中分离的量来进行 - 100毫克的小鼠骨骼肌，从而提供了以最小的材料的高通量。具有十分重要的意义，多井的能力耗氧量的技术来执行检测与数量时线粒体的最小的，结合经优化的分离方法，使研究人员进行大量的其他实验用的骨骼肌组织其余（例如，蛋白质印迹，RT-PCR法，酶测定法，等），这是通常与该动物模型的斗争。 总之，本文介绍的方法提供一步一步的指示，适用于使用量的小鼠骨骼肌最小的基于微孔板检测呼吸速率集合的性能。重要的是，所提出的方法需要大量的组织和线粒体最小。一旦掌握，本文中描述的技术将允许研究人员确定在一个常用的动物模型对线粒体耗氧量的化合物或基因产物的潜在机制。

骨骼肌线粒体的主要生理作用是从氧化磷酸化1产生ATP。重要的是，骨骼肌线粒体的运动能力2发挥具体作用，延缓衰老3，退行性疾病4和II型糖尿病5。作为老龄化社会，和II型糖尿病是死亡在美国6的 7 个首要原因，是否需要评估线粒体功能的方法已经变得越来越在生物医学研究7,8-更普遍。具体地讲，氧的消耗的测量在线粒体功能的评估呈实用，因为它代表的线粒体和核基因组之间的协调功能，表达氧化磷酸9的功能组件。 几种技术存在，使氧消耗在完整细胞的测定和隔离ð线粒体7-10。另外，测定中已经开发了用于多种细胞类型和组织，并在各种物种，其允许线粒体途径和呼吸控制9,11,12的测定。然而，目前在文献中的空隙在问候所有这些测定中使用分离线粒体从小鼠骨骼肌中基于微孔板氧消耗的技术使用的汇编。重要的是，利用基于微孔板透气性试验越来越多线粒体生物学家中并允许使用量的分离线粒体9的最小的高通量测量。因此，基于微孔板呼吸计测定的集合被开发，允许精确定位，其中异常和/或改编可以在电子传递链（ETC）来发生的。此外，两个附加的微孔板基于呼吸计测定法开发，使协调的betwee评估n中的三羧酸（TCA）循环和ETC，和β-氧化和ETC.之间重要的是，所提出的方法提供了一个简洁明了的方法来衡量线粒体功能的常用动物模型的机械变化。

<table><tbody><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>S7903</td><td>Store at RT</td></tr><tr><td>D-Mannitol</td><td>Sigma Aldrich</td><td>63559</td><td>Store at RT</td></tr><tr><td>Potassium phosphate monobasic, minimum 99.0%</td><td>Sigma Aldrich</td><td>P5379</td><td>Store at RT</td></tr><tr><td>Magnesium chloride hexahydrate, ACS reagent, 99.0-102.0%</td><td>Sigma Aldrich</td><td>M9272</td><td>Store at RT</td></tr><tr><td>HEPES minimum 99.5% titration</td><td>Sigma Aldrich</td><td>H3375</td><td>Store at RT</td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E4378</td><td>Store at RT</td></tr><tr><td>Essentially Fatty&amp;nbsp;</td><td>Sigma Aldrich</td><td>A6003</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Acid Free- BSA</td><td></td><td></td><td></td></tr><tr><td>Pyruvic Acid, 98%</td><td>Sigma Aldrich</td><td>107360</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Succinic Acid</td><td>Sigma Aldrich</td><td>S9512</td><td>Store at RT</td></tr><tr><td>L(-) Malic Acid, BioXtra, &amp;ge;95%</td><td>Sigma Aldrich</td><td>M6413</td><td>Store at RT</td></tr><tr><td>L-Glutamic acid</td><td>Sigma Aldrich</td><td>G1251</td><td>Store at RT</td></tr><tr><td>N,N,N&amp;prime;,N&amp;prime;-Tetramethyl-p-phenylenediamine</td><td>Sigma Aldrich</td><td>T7394</td><td>Store at RT</td></tr><tr><td>99%, powder [TMPD]</td><td></td><td></td><td></td></tr><tr><td>Palmitoyl L-carnitine chloride</td><td>Sigma Aldrich</td><td>P1645</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Oligomycin A, &amp;ge; 95% (HPLC)</td><td>Sigma Aldrich</td><td>75351</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Carbonyl cyanide 4-(trifluoromethoxy)</td><td>Sigma Aldrich</td><td>C2920</td><td>Store at 2 - 8 &amp;deg;C</td></tr><tr><td>phenylhydrazone</td><td></td><td></td><td></td></tr><tr><td>&amp;ge;98% (TLC), powder [FCCP]</td><td></td><td></td><td></td></tr><tr><td>Antimycin A from streptomyces sp.</td><td>Sigma Aldrich</td><td>A8674</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Adenosine 5&amp;prime;-diphosphate monopotassium salt dehydrate [ADP]</td><td>Sigma Aldrich</td><td>A5285</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Rotenone</td><td>Sigma Aldrich</td><td>R8875</td><td>Store at RT</td></tr><tr><td>Pierce&amp;trade; BCA Protein Assay Kit&amp;nbsp;</td><td>Thermo Scientific</td><td>23225</td><td>N/A</td></tr></tbody></table>

using isolated mitochondria from minimal quantities of mouse skeletal muscle for high throughput microplate respiratory measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

图1表示氧的消耗率（OCR）的丙酮酸/苹果酸盐，琥珀酸盐/鱼藤酮，棕榈酰肉碱/苹果酸盐，和谷氨酸/苹果酸测定法（偶联测定法）。这些测定法描记显示为耗氧率，或OCR，相对于时间，被背景correceted，并显示为点对点速率。每个小组代表氧消耗所描述的机遇与威廉姆斯14种不同的线粒体状态。第一面板代表基底耗氧量，或国家2.第二面板，注射的ADP后，表示最大连接呼吸，或国家3.第三面板，注射的寡霉素A（复合物V的抑制剂）后，表示呼吸因质子漏，或国家40。第四盘，注射FCCP后，表示极大的解偶联呼吸作用，或国家3U。最后，第五面板，注射抗霉素A的后，表示氧化呼吸的抑制。值得注意的是，人升线粒体状态有最小的标准偏差。这是由于在线粒体库存和线粒体/衬底混合物，和实现线粒体的粘附自旋（步骤2.6）后的单一单层的彻底混合。另一方面，装载不等线粒体在每个孔中，而不是实现线粒体的单个单层的微板的井引线显示在图2至增加标准偏差在每个状态。 在图1中显示的高原的描记为每个线粒体状态和为每个衬底。两个测量周期后达到的高原表明良好线粒体质量和线粒体保持整个测定法期间粘附到孔中。此外，实现鳍最大速率可能更希望的，因为这使研究人员取平均的OCR这些线粒体状态，从而减少可能发生的偏差通过任意选择点。 每孔线粒体的量通过优化试验来确定。每孔线粒体的最佳量应导致国家100-200皮摩尔/分钟/孔和状态3个评分&lt;之间2个评分1500皮摩尔/分钟/孔，因为这些值是多井氧的动态氧感应范围内消耗测量机。每孔中过多线粒体蛋白可能导致超出仪器（图3A）的动态范围同时进行文本识别。 图3示出了装载3.5每孔线粒体蛋白（蓝色曲线）的微克相比每孔加载线粒体蛋白的2.5微克（红跟踪）为琥珀酸/鱼藤酮测定法。载入每孔太多线粒体蛋白也可导致氧气的井的微室内用尽，从而防止OCR的精确测量对于每个连续测量9（图3B &lt;/ STRONG&gt;）。点对点的OCR是OCR的变化的瞬时速率。如果持平，OCR稳定/稳定，但如果减少，则有可能是一种生物或技术问题。在急剧下降，OCR在国3和国3U呼吸（ 图3A）是由排气测量（ 图3B）年底前供氧线粒体引起的。 图4表示的OCR与时间的电子流测定。跟踪校正并显示所描述的图1所示。在该试验中的第一个面板上表示丙酮酸/苹果酸盐州3U呼吸经由复一，第二面板，注射鱼藤酮后，表示抑制复合物I介导的呼吸。第三小组，注射琥珀后，代表基板刺激国家3U与电子进入ETC的复合物II（复合物II介导的呼吸）。第四盘，安迪美注射后霉素A，代表了抑制复合物III，因此总的呼吸。最后，第五面板，注射抗坏血酸/ TMPD后，代表复合物IV介导的呼吸。类似于图 1中的描记，所有线粒体状态有最小的标准偏差，并且每个速率有或几乎已经达到平台期。 <img alt="图1" src="/files/ftp_upload/53216/53216fig1.jpg" /> 图1.耦合测定。（A）中的10mM丙酮酸/ 5mM的苹果酸盐，（B）中的10mM琥珀酸/ 2μM鱼藤酮，（C）的 40μM棕榈肉碱/ 1mM的苹果酸盐， 和 （D）的10mM谷氨酸盐/ 10mM的苹果酸盐耦合线粒体呼吸描记法如通过氧消耗多井测量来确定。值表示为平均值±标准差。每孔装线粒体蛋白是3.5微克的所有检测，除了琥珀/腐烯酮，其利用2.5微克线粒体蛋白的每孔。数据表示n = 3的配对生物学重复。 OCR =耗氧率; ADP =二磷酸腺苷;寡=寡A; FCCP =羰基氰- 4 - （三氟甲氧基）苯腙;抗A =抗霉素A; PYR =丙酮酸; SUCC =琥珀酸; ROT =鱼藤酮; PAL-C =棕榈肉碱; GLUT =谷氨酸。 <a href="https://www.jove.com/files/ftp_upload/53216/53216fig1large.png" style="font-size: 14px; line-height: 28px;" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53216/53216fig2.jpg" /> 图2.高度可变丙酮酸/苹果酸含量。引起的不完全混合线粒体从线粒体库存在衬底/ MAS混合，从而导致可变线粒体亲高度可变的10mM丙酮酸/ 5mM的苹果酸盐法蛋白质装载在每口井。每孔装线粒体蛋白是3.5微克。数据表示n = 3的配对生物学重复。 OCR =耗氧率; ADP =二磷酸腺苷;寡=寡A; FCCP =羰基氰- 4 - （三氟甲氧基）苯腙;抗A =抗霉素A; PYR =丙酮酸。 <a href="https://www.jove.com/files/ftp_upload/53216/53216fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53216/53216fig3.jpg" /> 图3.重载线粒体蛋白为琥珀酸/鱼藤酮含量。 （A）引起的加载3.5每孔线粒体蛋白（蓝色跟踪）的微克的动态范围的多井氧消耗测量机之外氧消耗率相比每孔（红色曲线）线粒体蛋白的装载2.5微克。（乙）牛ygen张力接近零以下的ADP引起的加载每孔（蓝色曲线）过多的线粒体蛋白（3.5微克）FCCP注射相比，装载线粒体蛋白每孔的最佳量（2.5微克）（红色曲线）。数据代表N = 3成对生物元线粒体蛋白量的重复。 OCR =耗氧率; ADP =二磷酸腺苷;寡=寡A; FCCP =羰基氰- 4 - （三氟甲氧基）苯腙;抗A =抗霉素A; SUCC =琥珀酸; ROT =鱼藤酮; O 2 =氧;毫米汞柱=毫米汞柱。 <a href="https://www.jove.com/files/ftp_upload/53216/53216fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53216/53216fig4.jpg" /> 图4.电子流检测。5毫米丙酮酸/ 1毫米的苹果酸+ 4μMFCCP，电子˚F低线粒体呼吸测定跟踪如通过氧消耗多井测量来确定。值表示为平均值±标准差。每孔装线粒体蛋白是3.5微克。数据表示n = 3的配对生物学重复。 OCR =耗氧率;抗A =抗霉素A; ASC =抗坏血酸; TMPD = N，N，N&#39;，N&#39; -四甲基对苯二胺 。 <a href="https://www.jove.com/files/ftp_upload/53216/53216fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0"><tbody><tr><td> 试剂 </td><td> 股票集中度 </td><td> MW </td><td> 最终体积 </td><td> 大规模增加 </td><td> 评论/说明 </td></tr><tr><td></td><td> （M）的 </td><td> （克/摩尔） </td><td> （毫升） </strong&gt; </td><td> （g或毫升） </td><td></td></tr><tr><td> EGTA，pH 7.2的</td><td> 0.1 </td><td> 380.35 </td><td>百毫升1M的Tris基地</td><td> 3.801克</td><td>储存在4℃ </td></tr><tr><td> HEPES </td><td> 1 </td><td> 238.3 </td><td> 250毫升DIH 2 O的</td><td> 59.57克</td><td>储存在4℃ </td></tr><tr><td> 氯化镁 ，六</td><td> 1 </td><td> 203.31 </td><td> 250毫升DIH 2 O的</td><td> 50.82克</td><td>储存在4℃ </td></tr><tr><td>丙酮酸pH值7.4 </td><td> 0.1 </td><td> 88.06（配为14.11溶液） </td><td> 40毫升DIH 2 O的</td><td> 0.283 ml的丙酮酸</td><td>使1ml等份并储存在-20℃下，使新鲜每两周</td></tr><tr><td>琥珀酸PH值7.4 </td><td> 0.5 </td><td> 118.09 </td><td>百毫升DIH 2 O的</td><td> 9.4克苏ccinic酸</td><td>使1ml等份并储存在-20℃下</td></tr><tr><td>苹果酸，pH值7.4 </td><td> 0.5 </td><td> 134.09 </td><td>百毫升95％乙醇的</td><td> 6.7克苹果酸的</td><td>使200微升等分并储存在-20℃ </td></tr><tr><td> TMPD </td><td> 0.01 </td><td> 164.25 </td><td> 10毫升</td><td> 0.0164克</td><td>使300μl的等分试样并储存在-20℃;混合使用抗坏血酸，以保持TMPD的降低等摩尔量</td></tr><tr><td>棕榈左旋肉碱氯化物</td><td> 0.01 </td><td> 436.07 </td><td> 1.14 ml的95％乙醇的</td><td> 0.005克</td><td>使40微升等份并储存在-20℃ </td></tr><tr><td>寡á </td><td> 0.006 </td><td> 791.06 </td><td> 0.987 ml的95％乙醇的</td><td> 0.005克</td><td>使20微升等份并储存在-20℃ </td></TR&gt; <tr><td> FCCP </td><td> 0.01 </td><td> 254.17 </td><td> 3.9 ml的95％乙醇的</td><td> 0.01克</td><td>使40微升等份并储存在-20℃ </td></tr><tr><td>鱼藤酮</td><td> 0.001 </td><td> 394.4 </td><td> 10毫升95％乙醇的</td><td> 0.0039克</td><td>储存在-20℃ </td></tr><tr><td>抗霉素A </td><td> 0.005 </td><td> 548.63 </td><td> 9.12毫升95％乙醇的</td><td> 0.025克</td><td>储存在-20℃ </td></tr><tr><td> K + ADP </td><td> 0.5 </td><td> 501.32 </td><td> 3.9毫升DIH 2 O的</td><td> 1.0克</td><td>储存在-20℃ </td></tr><tr><td>苹果酸，pH值7.4 </td><td> 0.5 </td><td> 134.09 </td><td> 40毫升</td><td> 2.68克</td><td>使200微升等分并储存在-20℃ </td></tr></tbody></table><strong&gt;表1. 库存解决方案 <table border="0"><tbody><tr><td rowspan="2"> 试剂 </td><td> 股票集中度 </td><td> 大规模增加 </td><td rowspan="2"> 最后摩尔浓度/百分比 </td></tr><tr><td> （M）的 </td><td> （g或毫升） </td></tr><tr><td>蔗糖</td><td> - </td><td> 11.98克</td><td> 70毫米</td></tr><tr><td>甘露醇</td><td> - </td><td> 20.04克</td><td> 220毫米</td></tr><tr><td>磷酸二氢钾</td><td> - </td><td> 0.34克</td><td> 5毫米</td></tr><tr><td> 氯化镁 ，六</td><td> 1 </td><td> 2.5毫升</td><td> 5毫米</td></tr><tr><td> HEPES </td><td> 1 </td><td> 1.0毫升</td><td> 2毫米</td></tr><tr><td> EGTA </td><td> 0.1 </td><td> 5.0毫升</td><td> 1毫米</td></tr><tr><td>从本质上讲脂肪酸自由BSA </td><td> - </td><td> 1.0克</td><td> 0.20％ </td></tr></tbody></table> 表2. MAS混合pH值7.4 500毫升 :分装25毫升并储存在-20ºC*注:排除BSA的用于检测注射MAS组合。 <table border="0"><tbody><tr><td> 基材中 </td><td> 终浓度 </td><td> 库存量（微升） </td><td> MAS *量（毫升） </td></tr><tr><td rowspan="2">丙酮酸/马拉特</td><td rowspan="2"> 10毫米/ 10毫米</td><td>丙酮酸:1000 </td><td rowspan="2"> 9 </td></tr><tr><td>苹果酸:100 </td></tr><tr><td rowspan="2"> Succicinate /鱼藤酮</td><td rowspan="2">的10mM / 2μM </td><td>琥珀酸:200 </td><td rowspan="2"> 10 </td></tr><tr><td>鱼藤酮20 </td></tr><tr><td rowspan="3"> *丙酮酸/马拉特+ FCCP </td><td rowspan="3"> 5毫米/ 1毫米/ 4μM </td><td>丙酮酸:500 </td><td rowspan="3"> 10 </td></tr><tr><td> FCCP:4 </td></tr><tr><td>苹果酸:20 </td></tr><tr><td>棕榈左旋肉碱/马拉特</td><td> 40微米/ 1毫米</td><td> Palmitoylcarnitine:40马拉特:20 </td><td> 10 </td></tr><tr><td rowspan="2">谷氨酸/马拉特</td><td rowspan="2">的10mM / 10mM的</td><td>谷氨酸:400 </td><td rowspan="2"> 10 </td></tr><tr><td>苹果酸:200 </td></tr></tbody></table> 表3底物解pH为7.4:使新鲜的实验当天。 *电子流检测解决方案。 <table border="0"><tbody><tr><td> 注射中 </td><td> 集中 </td><td> 库存量（微升） </td> &lt;TD&gt; MAS的量（ml） </td><td> 注入墨盒数量 </td><td> 终浓度 </td></tr><tr><td></td><td></td><td></td><td></td><td></td><td> （注射板后） </td></tr><tr><td> ADP </td><td> 50毫米</td><td> 300微升</td><td> 3 </td><td> 50微升</td><td> 5.0毫米</td></tr><tr><td>寡á </td><td> 20μM </td><td> 10微升</td><td> 3 </td><td> 55微升</td><td> 2.0μM </td></tr><tr><td> FCCP </td><td> 40μM </td><td> 12微升</td><td> 3 </td><td> 60微升</td><td> 4.0μM </td></tr><tr><td>抗霉素A </td><td> 40μM </td><td> 24微升</td><td> 3 </td><td> 65微升</td><td> 4.0μM </td></tr></tbody></table> Ť能够4.注射剂再加测定pH值7.4:让新鲜的实验当天。 *偶联测定法包括（但不限于）丙酮酸/苹果酸盐，琥珀酸盐/鱼藤酮，棕榈酰肉碱/苹果酸盐，和谷氨酸/苹果酸盐。 <table border="0"><tbody><tr><td> 注射中 </td><td> 集中 </td><td> 库存量（g或UL） </td><td> MAS的量（ml） </td><td> 注入墨盒数量 </td><td> 终浓度（注射板后） </td></tr><tr><td>鱼藤酮</td><td> 20μM </td><td> 60微升</td><td> 3 </td><td> 50微升</td><td> 2.0μM </td></tr><tr><td>琥珀酸</td><td> 50毫米</td><td> 300微升</td><td> 3 </td><td> 55微升</td><td> 5.0毫米</td></tr><tr><td>抗霉素A </td><td> 40μM </td><td> 24μ; l </td><td> 3 </td><td> 60微升</td><td> 4.0μM </td></tr><tr><td> TMPD / Ascorabte </td><td> 1毫米，100毫米</td><td> TMPD:300微升</td><td> 3 </td><td> 65微升</td><td> 100μM的，10毫</td></tr><tr><td></td><td></td><td>抗坏血酸:0.059克</td><td></td><td></td><td></td></tr></tbody></table> 表5.注射剂的电子流测定 pH为7.4:使新鲜的实验当天<table border="0"><tbody><tr><td> 命令 </td><td> 时间（min） </td><td> ＃周期 </td></tr><tr><td>校准</td><td></td><td></td></tr><tr><td>等一下</td><td> 10分钟（以允许板从附着步骤暖） </td><td></td></tr><tr><td>拌</td><td> 1分钟</td><td rowspan="2"> 2 </td></tr><tr><td>我ASURE </td><td> 2分钟</td></tr><tr><td>注入</td><td></td><td></td></tr><tr><td>拌</td><td> 1分钟</td><td rowspan="2"> 2 </td></tr><tr><td>测量</td><td> 2分钟</td></tr><tr><td>注入乙</td><td></td><td></td></tr><tr><td>拌</td><td> 1分钟</td><td rowspan="2"> 2 </td></tr><tr><td>测量</td><td> 2分钟</td></tr><tr><td>注入Ç </td><td></td><td></td></tr><tr><td>拌</td><td> 1分钟</td><td rowspan="2"> 2 </td></tr><tr><td>测量</td><td> 2分钟</td></tr><tr><td>注入ð </td><td></td><td></td></tr><tr><td>拌</td><td> 1分钟</td><td rowspan="2"> 2 </td></tr><tr><td>测量</td><td> 2分钟</td></tr></tbody></table> 表6.仪运行协议。

Watch this Scientific Journal Video about Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements at JoVE.com

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

using-isolated-mitochondria-from-minimal-quantities-mouse-skeletal

Research

JoVE Journal

Developmental Biology

13.6K 次观看.  Virginia Tech. The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

视频: Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

发育生物学

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

通过注射心脏毒素急性骨骼肌再生的诱导

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

制备及文化活动从成年和老年人类骨骼肌成肌前体细胞/成肌细胞主要的

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

线粒体的制备及透气性评估，从骨骼肌组织通过穿刺活检获得的分离

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

医学

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

骨骼肌的线粒体分离

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

生物学

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 ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

从小鼠骨骼肌高通量微孔板呼吸测量最低数量的隔离线粒体

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

从小鼠骨骼肌中分离线粒体用于呼吸测定

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

<meta charset="utf8"></head><body>实时定量肌肉纤维中的线粒体呼吸

Measurement of Mitochondrial Respiration in Human and Mouse Skeletal Muscle Fibers by High-Resolution Respirometry

Mitochondrial function, a cornerstone of cellular energy production, is critical for maintaining metabolic homeostasis. Its dysfunction in skeletal muscle is linked to prevalent metabolic disorders (e.g., diabetes and obesity), muscular dystrophies, and sarcopenia. While there are many techniques to evaluate mitochondrial content and morphology, the hallmark method to assess mitochondrial function is the measurement of mitochondrial oxidative phosphorylation (OXPHOS) by respirometry. Quantification of mitochondrial OXPHOS provides insight into the efficiency of mitochondrial oxidative energy production and cellular bioenergetics. A high-resolution respirometer provides highly sensitive, robust measurements of mitochondrial OXPHOS in permeabilized muscle fibers by measuring real-time changes in mitochondrial oxygen consumption rate. The use of permeabilized muscle fibers, as opposed to isolated mitochondria, preserves mitochondrial networks, maintains mitochondrial membrane integrity, and ultimately allows for more physiologically relevant measurements. This system also allows for the measurement of fuel preference and metabolic flexibility - dynamic aspects of muscle energy metabolism. Here, we provide a comprehensive guide for mitochondrial OXPHOS measurements in human and mouse skeletal muscle fibers using a high-resolution respirometer. Skeletal muscle groups are composed of different fiber types that vary in their mitochondrial fuel preference and bioenergetics. Using a high-resolution respirometer, we describe methods for evaluating both aerobic glycolytic and fatty acid substrates to assess fuel preference and metabolic flexibility in a fiber-type-dependent manner. The protocol is versatile and applicable to both human and rodent muscle fibers. The goal is to enhance the reproducibility and accuracy of mitochondrial function assessments, which will improve our understanding of an organelle important to muscle health.

<meta charset="utf8"></head><body>作者聚焦:揭示代谢疾病中的线粒体功能和细胞代谢适应

Here, we describe a comprehensive method for measuring mitochondrial oxidative phosphorylation in fresh permeabilized skeletal muscle fibers from either human or mouse muscle. This method allows for the real-time quantification of mitochondrial respiration and the assessment of fuel preference and metabolic flexibility while preserving existing mitochondrial networks and membrane integrity.

通过高分辨率呼吸测定法测量人和小鼠骨骼肌纤维中的线粒体呼吸

Mitochondrial function, a cornerstone of cellular energy production, is critical for maintaining metabolic homeostasis. Its dysfunction in skeletal muscle ...