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蔗糖，10毫米的Na-HEPES，200μMEDTA，pH值7.4），并用清水冲洗干净血液。 </li></ol> 注意：为了获得最佳结果，线粒体缓冲液的pH值应进行调整，用KOH和醋酸的催化剂。 <ol start="3"><li>广场的心在一个寒冷的培养皿中，去除脂肪和心房，和肉细，使用刀片服务器，直到获得ING均匀的产品。 </li><li>剁碎心中转移到50ml Falcon管中，加入10毫升含0.1毫克/毫升胰蛋白酶及线粒体隔离缓冲允许样品消化在冰上10分钟。 </li></ol> 注：在分离过程中的胰蛋白酶分离线粒体和增加产量之间线粒体制剂应保持不变。我们建议，测试筹备分离的线粒体功能是胰蛋白酶的情况下，以确保酶的功能，不干扰。 <ol start="5"><li>线粒体分离缓冲液中加入10毫升的0.1％的脂肪酸​​游离BSA和5 mg胰蛋白酶抑制剂（大豆）和混合颠倒离心管数次中的胰蛋白酶。 </li></ol> 注：线粒体隔离缓冲可以预先准备的，并且可以被存储于-20℃下检查解冻后的pH值。胰蛋白酶，胰蛋白酶抑制剂或BSA必须是一个dded实验当天的线粒体隔离缓冲。 <ol start="6"><li>恢复消化组织由低到中速离心1分钟，在4°C </li><li>重悬组织在8毫升线粒体分离缓冲液用0.1％的脂肪酸​​BSA和同质化用聚四氟乙烯杵（4-6的通行证在1,200-14,00转）使用机动Dounce匀浆器。而同质化，保持样品在冰。 </li><li>在800xg转移入15ml Falcon管中，离心匀浆10分钟，在4℃下沉淀细胞核和组织碎片。 </li><li>含有线粒体到Eppendorf管中并离心，以8,000 xg离心15分钟，回收上清液在4℃下</li><li>小心删除顶白层，并用剩余的较深的颗粒在1毫升线粒体隔离缓冲中含有的线粒体。 </li><li>在8000xg离心15分钟，在4℃下弃上清，重悬线粒体颗粒在150微升线粒体伊索拉杂交缓冲液。查看线粒体进一步测定在冰上。 </li></ol> 注：为了达到最佳效果，在功能性检测，使用线粒体隔离4小时内。 <ol start="12"><li>量化线粒体蛋白，用Bradford法。 </li></ol> 2。分离线粒体的质量控制 2.1。测量呼吸控制率（RCR） <ol><li> ，校准Oxytherm氧电极用空气饱和水建立的最大氧气线。当氧得到一个稳定的水平，加入100微升新鲜制备的10mM的亚硫酸氢钠溶液建立零氧线。在30°C进行测量</li><li>加入2 ml线粒体呼吸用1μM鱼藤酮的Oxytherm室的缓冲液（125 mM KCl中的20mM HEPES，3毫摩尔的MgCl 2，400μMEGTA，300μMDTT，5mM的KH 2 PO 4，pH为7.2），密封腔室并启动收音丁。 </li><li>当氧信号是稳定的，添加250微克线粒体和记录的基础呼吸1分钟。在这一点上应该是非常低的呼吸，线粒体呼吸作用的内源性底物（呼吸状态1）。 </li><li>添加5mM的琥珀酸酯和1分钟的记录信号。耗氧量增加在状态2呼吸。 </li><li>加入150μMADP诱导状态3呼吸。在这一点上氧的消耗增加，因为通过氧化磷酸化合成ATP。记录信号，直到呼吸减慢，这表明的ADP消费和过渡到状态4呼吸。记录状态4呼吸至少1分钟。 </li><li>添加1μM的CCCP并记录信号，1分钟。非耦合的呼吸应该是最大的。 </li></ol> 注：CCCP线粒体呼吸的影响是剂量依赖性的高浓度的CCCP能抑制氧的消耗，因此应仔细吨itrated。 <ol start="7"><li>状态3呼吸期间由在状态4期间呼吸的氧消耗率除以氧消耗率计算呼吸控制率（RCR）。的目标RCR应≥4。 </li></ol> 2.2。评估线粒体膜的完整性（细胞色素C测试） <ol><li>彻底洗净Oxytherm的腔室用70％乙醇和水，并重复步骤2.1.2 -2.1.4。 </li><li>加入1 mM的ADP和记录氧气消耗为1分钟。 </li><li>添加10μM细胞色素c。由于细胞色素c的完整的线粒体膜是不可渗透的，呼吸增加表示断裂或损坏的线粒体膜。 </li></ol> 3。测量的线粒体通透性转换孔开放<ol><li>设置荧光光谱仪，多参数测量的mtPTP开放。使用FelixGx程序，创建一个新的硬件配置（广达主稳步站特），其包括光源，激发单色器，样品室和两个检测器的激发单色器（ 图4）相对于定位在90°和270°。为了达到最大的时间分辨率，发射波长（90°和270°）必须被禁止，排放过滤器的样品室，在90°和270°的硬件配置菜单中启用。此步骤将删除的发射波长的电机控制设备，每个单色器和固定在一个特定的波长。如果没有被禁用的发射波长，每个探测器将两个波长之间的循环和重复测量将被记录下来。 </li><li>创建一个新的收购协议用染料类型多，并输入以下染料： <ul><li>的Fura（高Ca + +）：激发340nm，发射波长525 nm（检测器1） </li><li>的Fura（低Ca + +）：激发380nm的发射检测器1（525纳米） </li><li> JC1（总）：激发543 nm的，发射波长595nm（检测器） </li><li> JC1（单体）：激发498 nm处，发射波长525 nm（检测器1） </li><li>肿胀：激发波长525 nm，发射波长525 nm（检测器） </li></ul></li><li>设置的激发和发射狭缝为1 nm至37℃的温度</li><li>要获得比例的Fura和JC-1信号，产生两个派生的痕迹：的Fura（高Ca + +）/的Fura（低Ca + +）和：JC1（合计）/ JC1（单体）。 </li><li>手动调整检测器1和2到525 nm和595nm处的发射波长，分别。 </li><li>线粒体混合1毫升分析缓冲液（120毫摩尔NaCl，10 mM KCl中，1mM的KH 2 PO 4，20mM的HEPES的Tris，pH值7.2），鱼藤酮，用1μM5mM的琥珀酸和800纳米的Fura FF在一次性4双面甲基丙烯酸甲酯试管中。添加250微克线粒体和地方样品在样品室。确保磁力搅拌器接通。 </li><li>开始记录。 1分钟后，暂停收购，加入500 nM的JC-1，然后重新启动一个cquisition。 JC-1的比例信号应该增加，说明染料的吸收活跃的线粒体。继续录制JC-1“信号，直到已达到了高原（通常在5分钟内）。 </li><li>添加20μM 氯化钙 。应遵守的Fura FF比信号的瞬时增加，所增加的Ca 2 +在检测缓冲液，其次是由于线粒体Ca 2 +摄取逐渐减少。 JC-1的比例信号，应表现出短暂下降表示轻微的线粒体膜去极化。 </li><li>等待，直到基础水平之前，除了另一氯化钙脉冲（1-1.5分）的Fura FF比信号返回。继续脉冲以固定的时间间隔，直到线粒体不能积累的Ca 2 +和开始到测定缓冲液释放Ca 2 +的 。 mtPTP开口是可视化的同时增加的Fura FF比信号由于对Ca 2 +释放的JC-1信号的比率，减少由于膜POTE周期边值 ​​问题PBVP崩溃，而且在光散射减少由于线粒体肿胀（ 图5）。 </li><li> mtPTP激活之后，验证的Fura FF，JC-1和肿胀信号的特异性分别为1mM的EGTA，1μMCCCP和5微克/毫升丙甲菌素。 </li><li>为了验证特异性的mtPTP开放，重复步骤3.6 - 3.9中存在的1μM的亲环蛋白D抑制环孢素A（亲环素D是一个组件的mtPTP）。在环孢霉素A，开放的mtPTP需要明显更氯化钙的2个脉冲比对照样品（ 图6）。 </li></ol> 4。代表性的成果 图2显示了一个典型的小鼠离体心脏线粒体呼吸控制。状态3呼吸是通过加入ADP线粒体呼吸的上琥珀酸，由氧的消耗显着增加，其特征在于，相对于向substra德孤单。加入ADP启动状态，在此期间，呼吸耗氧量的放缓，是媲美率达到之前ADP此外耗尽。 RCR是通过将状态3呼吸的状态4呼吸的氧气消耗率。细胞色素 c的测试用于测定线粒体膜外的完整性：细胞色素 c被添加到线粒体呼吸的琥珀酸和ADP，观察不再增加呼吸时，表示一个完整的外线粒体膜（ 图3）。 如图4所示，在离体心脏线粒体的mtPTP开放的代表图像。在这种情况下，通过加入4 CaCl 2的脉冲（20μM）在1.5分钟的间隔。触发mtPTP开口几乎同时发布的线粒体Ca 2 +（增加的Fura FF比信号），氧代谢的崩溃是明显的mtPTP开幕里亚尔膜电位（减少JC-1比信号）和增加线粒体体积（减少溶胀信号）。当环孢素A被添加，其中结合到亲环素D成分mtPTP，mtPTP开口需要7个脉冲的CaCl 2，而不是4（ 图5）。 <img alt="图1" src="/files/ftp_upload/4131/4131fig1.jpg" /> 图1。的多参数线粒体通透性转换孔开放的协议离体心脏线粒体的流程图。心是收获的小鼠，并通过差速离心分离线粒体。线粒体制剂然后定性评估用极谱测量中存在的细胞色素 c的呼吸控制比和线粒体膜完好。在分离线粒体线粒体通透性转换孔开放所引发的顺序的CaCL 2的补充和与一个荧光分光光度计测定监察释放Ca 2 +从线粒体膜电位崩溃和肿胀的线粒体基质中。 <img alt="图2" src="/files/ftp_upload/4131/4131fig2.jpg" /> 图2的测量 离体心脏线粒体呼吸控制率（RCR）。离体心脏线粒体氧消耗期间测量状态3呼吸琥珀酸酯和ADP的存在下，和在状态4期间呼吸后ADP消费。分离线粒体解偶联剂的反应，测定由加入CCCP。图表上的数字是在纳摩尔/分钟/ mg蛋白分离的线粒体的氧消耗率。 <img alt="图3" src="/files/ftp_upload/4131/4131fig3.jpg" /> 图3测量<html>的离体心脏线粒体膜的完整性。线粒体膜的完整性被确定琥珀酸盐的存在下，和ADP和之后的细胞色素 c的此外，通过测量氧的消耗，在分离的线粒体。图表上的数字是在纳摩尔/分钟/ mg蛋白分离的线粒体的氧消耗率。 <img alt="图4" src="/files/ftp_upload/4131/4131fig4.jpg" /> 图4。荧光光谱仪的硬件配置为多参数测量的mtPTP开幕。的硬件配置的荧光分光光度计，包括光源，激发单色器，样品室和两个检测器，与固定发射波长在525nm和595 nm。检测器1被用于信号检测的Fura FF高和低的Ca 2 +，JC-1单体和溶胀信号。探测器测量JC-1总的信号。 jove_content的“Fo：保持together.within”页面=“总是”&gt; <img alt="图5" src="/files/ftp_upload/4131/4131fig5.jpg" /> 图5。多参数在离体心脏线粒体测量的mtPTP开幕。所引发的顺序增加20μM 氯化钙和mtPTP开放为特征的Ca 2 +释放的线粒体，线粒体膜电位崩溃，增加线粒体基质体积（肿胀）。线粒体外的Ca 2 +的比例指标的Fura FF（绿线）和JC-1（红色线）和膜电位的测量。线粒体肿胀记录（黑迹线）为525 nm的光散射测定。 JC-1的特异性和肿胀信号进行了测试用1μMCCCP和5微克/毫升丙甲菌素（微生物毒素）。 <img alt="图6" src="/files/ftp_upload/4131/4131fig6.jpg" /> 图6的多参数测量的mtPTP开在离体心脏线粒体中存在的环孢素A环孢素A（1μM）CaCl 2的脉冲需要，打开mtPTP在离体心脏线粒体的数量增加。

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没有利益冲突的声明。

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

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 ...

multi-parameter-measurement-permeability-transition-pore-opening

Research

JoVE Journal

Biology

21.4K 次观看.  University of Washington, Seattle. 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 2 +

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

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

高度耦合的大鼠心肌线粒体的制备

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

科研

生物学

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

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

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

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

单线粒体超氧闪烁在完整心脏或共聚焦成像<em&gt;在体内</em

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

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

在离体血液灌注大鼠肺备量化单微血管通透性

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

测量渗透水渗透系数（P<sub&gt; F</sub&gt;）球形细胞：隔离植物原生质体为例。

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

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.

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 ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

平滑肌功能中分离出来的组织浴，应用药理学研究测量

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

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 ...

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of myocardial metabolism have been linked to the development of contractile dysfunction and heart failure. Therefore, unraveling the link between metabolism and contraction should shed light on some of the mechanisms governing cardiac adaptation or maladaptation in disease states. The isolated working rat heart preparation can be used to follow, simultaneously and in real time, cardiac contractile function and flux of energy providing substrates into oxidative metabolic pathways. The present protocol aims to provide a detailed description of the methods used in the preparation and utilization of buffers for the quantitative measurement of the rates of oxidation for glucose and fatty acids, the main energy providing substrates of the heart. The methods used for sample analysis and data interpretation are also discussed. In brief, the technique is based on the supply of 14C- radiolabeled glucose and a 3H- radiolabeled long-chain fatty acid to an ex vivo beating heart via normothermic crystalloid perfusion. 14CO2 and 3H2O, end byproducts of the enzymatic reactions involved in the utilization of these energy providing substrates, are then quantitatively recovered from the coronary effluent. With knowledge of the specific activity of the radiolabeled substrates used, it is then possible to individually quantitate the flux of glucose and fatty acid in the oxidation pathways. Contractile function of the isolated heart can be determined in parallel with the appropriate recording equipment and directly correlated to metabolic flux values. The technique is extremely useful to study the metabolism/contraction relationship in response to various stress conditions such as alterations in pre and after load and ischemia, a drug or a circulating factor, or following the alteration in the expression of a gene product.

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

为葡萄糖和脂肪酸氧化率在大鼠离体的测定方法

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

鼠标离体肾技术

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found responsible for very diverse diseases, ranging from neurological pathologies to cancer. Interestingly, mitochondria have recently been shown to display the capacity to be transferred between cell types, notably from human mesenchymal stem cells (MSC) to cancer cells in coculture conditions, with metabolic and functional consequences for the mitochondria recipient cells, further enhancing the current interest for the biological properties of these organelles.
Evaluating the effects of the transferred MSC mitochondria in the target cells is of primary importance to understand the biological outcome of such cell-cell interactions. The MitoCeption protocol described here allows the transfer of the mitochondria isolated beforehand from the donor cells to the target cells, using MSC mitochondria and glioblastoma stem cells (GSC) as a model system. This protocol has previously been used to transfer mitochondria, isolated from MSCs, to adherent MDA-MB-231 cancer cells. This mitochondria transfer protocol is adapted here for GSCs that present the specific particularity of growing as neurospheres in vitro. The transfer of the isolated mitochondria can be followed by fluorescence-activated cell sorting (FACS) and confocal imaging using mitochondria vital dyes. The use of mitochondria donor and target cells with distinct haplotypes (SNPs) also allows detection of the transferred mitochondria based on the concentration of their circular mitochondrial DNA (mtDNA) in the target cells. Once the protocol has been validated with these criteria, the cells harboring the transferred mitochondria can be further analyzed to determine the effects of the exogenous mitochondria on biological properties such as cell metabolism, plasticity, proliferation and response to therapy.

Here, a protocol (MitoCeption) is presented to transfer mitochondria, isolated from human mesenchymal stem cells (MSC), to glioblastoma stem cells (GSC), with the goal of studying their biological effects on GSC metabolism and functions. A similar protocol can be adapted to transfer mitochondria between other cell types.