Name: multi-parameter measurement of the permeability transition pore opening in isolated mouse heart mitochondria
Uploaded: 2012-09-07T12:00:00
Description: Watch this Scientific Journal Video about Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria at JoVE.com

这项工作得到了HL094536（BJH）。

1。从小鼠心脏线粒体的分离<ol><li>分离心脏线粒体，麻醉和牺牲小鼠，根据您的当地机构动物管理和使用委员会批准的程序。 </li></ol> 注：线粒体隔离协议的所有步骤必须执行在冰上。用冰冷的缓冲和预冷的培养皿中，Falcon管和Eppendorf管。在协议中给出的体积的样品中含有2小鼠心脏。 <ol start="2"><li>删除的心，把他们在寒冷的线粒体分离缓冲液（300 mM蔗?...

<ol>
	<li>Kroemer, G., Galluzzi, L., Brenner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Membrane+Permeabilization+in+Cell+Death.">Mitochondrial Membrane Permeabilization in Cell Death.</a> Physiol. Rev. 87, 99-163 (2007).</li><li>Elrod, J., Wong, R., Mishra, S., Vagnozzi, R. J., Sakthievel, B., Goonasekera, S. A., Karch, J., Gabel, S., Farber, J., Force, T., Brown, J. H., Murphy, E., Molkentin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclophilin+D+controls+mitochondrial+pore-dependent+Ca2++exchange,+metabolic+flexibility,+and+propensity+for+heart+failure+in+mice.">Cyclophilin D controls mitochondrial pore-dependent Ca2+ exchange, metabolic flexibility, and propensity for heart failure in mice.</a> J. Clin. Invest. 120, 3680-3687 (2010).</li><li>Hom, J. R., Quintanilla, R. 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L., O'Rourke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Ca2++influx+and+efflux+rates+in+guinea+pig+cardiac+mitochondria:+low+and+high+affinity+effects+of+cyclosporine+A.">Mitochondrial Ca2+ influx and efflux rates in guinea pig cardiac mitochondria: low and high affinity effects of cyclosporine A.</a> Biochim. Biophys. Acta. 1813, 1373-1381 (2011).</li><li>Saks, V. A., Kuznetsov, A. V., Kupriyanov, V. V., Miceli, M. V., Jacobus, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creatine+kinase+of+rat+heart+mitochondria.+The+demonstration+of+functional+coupling+to+oxidative+phosphorylation+in+an+inner+membrane-matrix+preparation.">Creatine kinase of rat heart mitochondria. 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这里介绍的协议描述了必要的实验步骤隔离小鼠心脏线粒体通透性转换孔开放，以评估在离体心脏线粒体（ 图1和图4）：过程，呼吸控制，确保其完整性和功能，线粒体期间监测参数mtPTP开放和他们的测量采用的染料，成立荧光分析仪器，表征mtPTP开幕。在本协议中的荧光分光光度计采用允许同时这三个参数的快速测量。然而，可以采用使用其它市售仪器的基本协议，尽?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>胰蛋白酶</td><td> Sigma-Aldrich公司</td><td> T3030 </td></tr><tr><td>胰蛋白酶抑制剂（大豆） </td><td> Sigma-Aldrich公司</td><td> T9128 </td></tr><tr><td>保险粉</td><td> Sigma-Aldrich公司</td><td> 71699 </td></tr><tr><td>鱼藤酮</td><td> Sigma-Aldrich公司</td><td> R8875 </td></tr><tr><td>细胞色素 c </td><td> Sigma-Aldrich公司</td><td> C7752 </td></tr><tr><td>丙甲菌素</td><td> Sigma-Aldrich公司</td><td> A4665 </td></tr><tr><td> CCCP </td><td> Sigma-Aldrich公司</td><td> C2759 </td></tr><tr><td>环孢素A </td><td> Calbiochem公司</td><td> 239835 </td></tr><tr><td>呋喃FF </td><td> Invitrogen公司</td><td> F14180 </td></tr><tr> &lt;TD&gt; JC-1 </td><td> Invitrogen公司</td><td> T3168 </td></tr><tr><td>组织磨床波特埃尔维耶姆特富龙杵15毫升</td><td>惠顿实业</td><td></td></tr><tr><td>架空搅拌器</td><td>惠顿实业</td><td> 903475 </td></tr><tr><td> oxytherm（温度控制氧电极） </td><td> Hansatech工具</td><td></td></tr><tr><td> QuantaMaster 80双发射荧光分光光度计</td><td>光子技术国际公司</td><td></td></tr></tbody></table>

multi-parameter measurement of the permeability transition pore opening in isolated mouse heart mitochondria

线粒体通透性转换孔（mtPTP）是一种非特异性通道，形成内线粒体膜的运输溶质的相对分子​​质量小于1.5 kDa的。虽然最终分子身份的孔隙仍在辩论，如亲环蛋白D，VDAC和ANT的蛋白质有助于mtPTP形成。虽然参与的mtPTP开放的细胞死亡完善，越来越多的证据表明，mtPTP供应的生理作用过程中线粒体Ca 2 +稳态，生物能和氧化还原信号3。 被触发mtPTP开口矩阵的Ca 2 +，但其活性可以是由多个其它因素，如氧化应激，腺嘌呤核苷酸耗尽，高浓度的Pi，线粒体膜去极化或解偶联，和长链脂肪酸的4调制。 在体外 ，mtPTP开口可以ACHieved线粒体基质内Ca 2 +浓度增加，通过外源添加的Ca 2 +（钙保持能力）。当Ca 2 +的水平内线粒体达到一定的阈值，mtPTP打开和促进的Ca 2 +释放的质子动力，耗散，膜电位崩溃和线粒体基质体积（肿胀）的增加，最终导致破裂的线粒体外膜和细胞器的功能不可逆转的丧失。 在这里，我们描述了一种荧光检测，允许进行全面的表征mtPTP开离体小鼠心脏线粒体。该法涉及到3线粒体发生改变时mtPTP开幕的参数，同时测量：线粒体Ca 2 +处理（吸收和释放的Ca 2 +浓度测定介质中），线粒体膜电位，并mitochondrial量。采用的染料为Ca 2 +的测定培养基及线粒体膜电位的测量的Fura FF，膜不能渗透的比例的指标，进行中的Ca 2 +和JC-1，阳离子的存在下，在激发波长的移位，形成绿色的单体或红色总量的比例指标，分别在低和高的膜电位，。线粒体量的变化记录的线粒体悬浮液的光散射测量。由于高品质，功能性线粒体所需要的的mtPTP开放试验，我们还描述了必要的步骤，以获得完整的，高度耦合和功能的离体心脏线粒体。

Watch this Scientific Journal Video about Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria at JoVE.com

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria (Video) | JoVE

这里介绍一个荧光光谱的测量小鼠离体心脏线粒体中的线粒体通透性转换孔开放的协议。该法涉及同时测定线粒体钙<sup&gt; 2 +</sup处理，线粒体膜电位和线粒体体积。获得高品质和功能性的心脏线粒体的程序也被描述。

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 ...

Research

JoVE Journal

Biology

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

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