这项工作得到了国家衰老研究所（AG04418），国家药物滥用研究所，国立卫生卡罗林斯卡医学院研究生院合作计划研究院，卡罗林斯卡医学院，瑞典研究理事会，瑞典脑动力，和瑞典的脑基金会。非常感谢马蒂亚斯Karlen博士和朱Coppotelli与图1和2，分别创造性的支持;卡琳Pernold技术援助和Drs。巴里J. Hoffer，拉尔斯奥尔森和尼尔斯戈兰拉尔森许多有益的建议和讨论。

1。组织编制为cryosectioning <ol><li>按照与现有的伦理许可证牺牲的动物，无论是颈椎脱位或斩首。 </li><li>快速收集利益的组织（如大脑），并迅速冻结干冰（组织可能需要用液态氮冷冻，以获得最佳的形态在戊烷或丙烷冻结）。铝箔存储于-80℃直至组织准备节。 </li><li> cryosectioning冰冻组织嵌入在准备。 </li><li>收集14微米的低温恒温器部分在-21 ° C（可能需要调整温度± 1-2 ° C）。解冻部分上使用coverslipping直到准备使用在-20 ° C加热块，并将其存储幻灯片的幻灯片。 </li></ol> 2。环氧合酶组织化学<ol><li>让幻灯片在室温下干燥1小时。将幻灯片在幻灯片用湿滤纸染色室，切成条状。为了获得consisten在每个实验吨结果，建议处理最多10％实验幻灯片，以尽量减少时间延误。 </li><li>准备下化学罩1X民建联，100微米0.1 M的PBS pH值= 7.0细胞色素C。涡迅速。 </li><li>新增2微克牛过氧化氢 ​​酶（2微克毫升 -1或-1毫升约4 IU）。震荡向上突破所有的过氧化氢酶颗粒混合。 </li><li>应用150 - 200μL孵化中期到每个幻灯片，使用枪头，均匀地分布到所有路段。 </li><li> 40分钟的幻灯片在37 ° C。 </li><li>从幻灯片中删除多余的解决方案。洗净幻灯片4次，每次10分钟，在0.1 M的PBS pH值= 7.0。 </li><li>返回幻灯片，幻灯片染色室，用湿纸条。 </li></ol> 3。 SDH组织化学<ol><li>准备下一个化学罩1.5毫米NBT，琥珀酸钠130毫米，0.2毫米的PMS，叠氮化钠在0.1 M的PBS pH值= 7.0和1.0 mm。注意屏蔽PMS的由轻。涡迅速。 </li><li> 150-200μL孵化介质应用到每个幻灯片，使用枪头，均匀地分布到所有路段。 </li><li> 40分钟的幻灯片在37 ° C。 </li><li>从幻灯片中删除多余的解决方案。洗净幻灯片4次，每次10分钟，在0.1 M的PBS pH值= 7.0。 </li><li>在以下浓度的乙醇脱水为2分钟的幻灯片：70％，70％，95％，95％，99.5％。然后让一个额外的99.5％步骤10分钟。 </li><li>广场幻灯片二甲苯10分钟。山与Entellan和盖玻片。允许的幻灯片干燥通风的地方过夜，或至少1-2小时。 </li></ol> 4。线粒体功能障碍的测定<ol><li>线粒体功能障碍的金额是由蓝染色细胞的数量表示。半量化这些款项，幻灯片应编码和明场显微镜下观察。应该盲目进行半定量<html>规模的基础上使用，例如从0-4（0，没有蓝染色; 4，只有蓝染色）。最好是从一个给定的主题/动物的几个部分执行这种半定量计算出每个主题/动物的平均值。 </li><li>统计应使用非参数检验，如曼 - 惠特尼或克鲁斯卡尔 - 沃利斯。 </li></ol> 5。适当的特异性控制<ol><li>特异性COX活性控制，重复“考克斯组织化学”的步骤，并添加2.5毫米的叠氮化钠，终端呼吸链抑制剂。 </li><li>对于SDH活性的特异性控制，重复“的SDH组织化学步骤，”琥珀酸钠的去除和另外50毫米丙二酸，SDH的竞争性抑制剂。 </li><li>洗涤和脱水乙醇系列中的一个部分，然后装入和盖玻片步骤3.4中所述的幻灯片 - 3.6。 </li></ol> 6。代表性的成果： 帐篷“的总体方案&gt;的适当的COX / SDH双标从野生型和过早衰老线粒体DNA的mutator小鼠的大脑切片组织化学的COX / SDH双标签组织化学检测，如图2所示。代表性的例子显示在图3暗棕黄色染色（图3，左图）在野生型小鼠表现出正常的COX活性细胞呼吸链指出不足之处，由蓝染色，在12周大的线粒体DNA的mutator小鼠显示，这些不足之处成为线粒体DNA的mutator岁至46周（图3，中心和右侧面板）小鼠更广泛。 不适当的COX / SDH双标记野生型小鼠的大脑由于不足的COX标签部分的例子如图4所示。 COX活性的示范孵化时间不足，或减少coverslipping孵化过程中的幻灯片，氧分子的可用性，导致在民建联的反应减少沉积离子产品，从而形成蓝色甲臜最终产品允许在SDH孵化。 COX和SDH活动也可以被单独调查（图5，左，中心）;然而，连续的标签是有助于识别与COX缺陷的细胞，由于在SDH孵化形成的蓝色沉淀（图3，中心和右）。特异性COX和SDH活动的控制，也可以做（图5，右）。 <img alt="图1" src="/files/ftp_upload/3266/3266fig1.jpg" /> 图1。线粒体呼吸配合物四 。线粒体呼吸链位于内膜，包括五个复合物。呼吸链的目的是从复杂的运输电子I至IV和这样做，它创建了一个跨内膜复杂V（ATP酶）用于产生ATP的质子梯度。 represe红色六边形新台币亚基由线粒体DNA编码。白色的六边形代表由核DNA（注意是完全的核基因组编码的复杂II）编码的亚基。因此，在线粒体基因组的突变可能会导致呼吸链功能障碍由于呼吸链复合物的亚基突变。 <img alt="图2" src="/files/ftp_upload/3266/3266fig2.jpg" /> 图2的COX / SDH双标组织化学检测流程图 。解剖感兴趣的器官，迅速冻结干冰组织，并存储他们在 - 80 ° C。收集低温恒温器的部分，并保持在 - 20℃直至使用。允许在室温下风干节1小时。孵化介质准备的COX组织化学，应用幻灯片，和40分钟在37 ° C。 PBS中4次，每次洗10分钟，洗净的部分。准备用于SDH组织化学孵化介质，适用于它的SLIDES，并再次在37 ° C孵育40分钟再次在PBS清洗部分，在乙醇系列脱水，然后装入和盖玻片的幻灯片。准备明场显微镜下查看1-2个小时内的COX / SDH双标部分。 <img alt="图3" src="/files/ftp_upload/3266/3266fig3.jpg" /> 图3的COX / SDH双标代表性的例子 。从野生型和过早衰老线粒体DNA的mutator小鼠的大脑切片顺序标示COX和SDH活动。 （比例尺：200微米）普通COX活性（深褐色表示）是从野生型小鼠的海马（左）所示。 COX缺陷（以蓝色表示），揭示了从线粒体的mutator小鼠的海马区（中心和右）。有mtDNA中的mutator小鼠的年龄由46个星期在COX活性的进一步下降，这表明呼吸链功能障碍的广泛发作。观察线粒体在线粒体DNA的mutator小鼠12线粒体功能障碍是引起mtDNA的点突变，以及增加5线性缺失水平高的水平。 <img alt="图4" src="/files/ftp_upload/3266/3266fig4.jpg" /> 图4。不适当的COX / SDH双标签的例子。从野生型小鼠的大脑切片顺序标示COX和SDH活动。 （比例尺：200微米）的孵育时间不足COX活性的示范（10和25分钟），在减少褐色DAB反应产物沉积，相比到40分钟的孵育时间（左边和中间的）。允许的蓝色甲臜最终产品的过程中形成的SDH孵化缩短孵化时间，误导性暗示与COX缺陷细胞的存在。 Coverslipping在COX孵化的幻灯片，也导致不准确的形成和沉积民建联的反应离子产品（右）。 <img alt="图5" src="/files/ftp_upload/3266/3266fig5.jpg" /> 图5，我ndividual COX和SDH的标签和特异性控制 。来自野生型小鼠的脑切片分别标示为深褐色和蓝色，分别为（左边和中间的）表示COX和SDH活动，。虽然COX和SDH活动可以单独标签，连续标签已被证明是定位与细胞线粒体功能障碍的优势。为特异性COX和SDH活动，从野生型小鼠在大脑控制的一个例子显示的标签的情况下（右）。 （比例尺：200微米）。

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没有利益冲突的声明。

合并COX / SDH组织化学方法使细胞线粒体功能障碍的可视化。这种技术，与早期的研究可追溯到到1968年，仍然深受市民欢迎，许多考虑它的“金标准”，确定线粒体疾病患者14,19,26,27。现在常用的调查mtDNA突变驱动的老化和衰老相关的疾病 12,13,18,20,21,24 。 COX / SDH双标记方法经常被用来与其他技术的同时，确定具体的mtDNA突变，并进一步调查，如oximetric测量和分光光度酶分析2...

<table border="1"><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>干冰</td><td> AGA气体AB </td><td>块形式</td><td></td></tr><tr><td>戊烷（2 - 甲基） </td><td> Sigma - Aldrich公司</td><td> 277258 CAS：78-78-4 </td><td></td></tr><tr><td> Cyrostat嵌入解决方案</td><td>樱花Finetek </td><td>组织TEK 4583 </td><td></td></tr><tr><td>低温恒温器</td><td> MICROM </td><td> MICROM型号HM 500M </td><td></td></tr><tr><td>幻灯片</td><td> Thermo Scientific的</td><td>超级冰霜加 门泽尔格拉泽 J1800AMWZ </td><td></td></tr><tr><td>盖玻片 硼硅玻璃</td><td> VWR International的</td><td> 16004-098 </td><td> 24 × 50 mm的</td></tr><tr><td>滤纸</td><td> Munktell过滤器AB </td><td>质量：1350 文章编号：242 001 </td><td> 430 × 430毫米</td></tr><tr><td> 3,3&#39; - 二氨基tetrahydrochloride（DAB） </td><td> Sigma - Aldrich公司</td><td>西格玛液体基底系统，D7304 </td><td></td></tr><tr><td>细胞色素 C（III型，从马的心脏） </td><td> Sigma - Aldrich公司</td><td> C2506 CAS：9007-43-6 </td><td></td></tr><tr><td>牛过氧化氢酶（肝脏） </td><td> Sigma - Aldrich公司</td><td> C9322 CAS：9001-05-2 </td><td></td></tr><tr><td>硝基四氮唑（NBT） </td><td> Sigma - Aldrich公司</td><td> N6876 CAS：298-83-9 </td><td></td></tr><tr><td>琥珀酸钠</td><td> Sigma - Aldrich公司</td><td> S2378 CAS：6106-21-4 </td><td></td></tr><tr><td> methosulfate吩嗪（PMS） </td><td> Sigma - Aldrich公司</td><td> P9625 CAS：299-11-6 </td><td> PMS是光敏感。免受光线照射。 </td></tr><tr><td>叠氮化钠</td><td> Sigma - Aldrich公司</td><td> S8032 CAS：26628-22-8 </td><td></td></tr><tR&gt; <td>二甲苯</td><td> VWR International的</td><td> EM - XX0060 - 4 </td><td></td></tr><tr><td> Entellan </td><td> VWR International的</td><td> 100503-870 </td><td></td></tr><tr><td>丙二 （丙二酸） </td><td> Sigma - Aldrich公司</td><td> M1296 CAS：141-82-2 </td><td></td></tr></table>

visualization of mitochondrial respiratory function using cytochrome c oxidase / succinate dehydrogenase (cox/sdh) double-labeling histochemistry

线粒体DNA（mtDNA）的缺陷是疾病的重要原因，可能背后的老龄化和老龄化有关的改建1,2。衰老的线粒体理论表明mtDNA突变的作用，它可以改变生物能动态平衡和细胞的功能，在衰老过程中 3 。财富已编译的证据支持这一理论的1,4，例如线粒体DNA的mutator鼠标5;然而，线粒体老化损坏的确切作用是不完全了解6,7。 观察呼吸酶的活性，是一个简单的方法进行调查线粒体功能障碍。复杂的四，或细胞色素 c氧化酶（COX），是对线粒体功能至关重要。环氧合酶的催化亚基由线粒体DNA编码和复杂的装配（图1）是必不可少的。因此，适当的合成和功能主要是基于对线粒体DNA的完整性 2 。虽然可以调查其他呼吸道疾病复合物，复合物IV和II是最适合组织化学检查 8,9 。复杂的二，或琥珀酸脱氢酶（SDH），完全是由核DNA（图1）编码，其活动通常不会受损线粒体的影响，虽然增加可能表明线粒体生物合成10-12。观察受损的线粒体DNA在线粒体疾病，老化，与年龄有关的疾病往往导致低或缺乏COX活性2,12-14细胞的存在。虽然COX和SDH活动可以单独进行调查，15,16连续的双标记方法已被证明是在寻找与细胞线粒体功能障碍 12,17-21有利。 许多检测的最佳宪法已经确定，如底物浓度，电子受体/捐助者，中间电子载体，pH值的影响，反应吨IME 9,22,23。 3,3&#39; -二氨基联苯胺（DAB）是一种有效和可靠的电子供体 22 。在运作的COX细胞，棕色indamine聚合物产品本地化线粒体嵴和饱和细胞22。因此，这些细胞功能失调的COX将不饱和民建联产品，硝基四氮唑（NBT），电子受体的减少SDH活性的可视化，一个蓝色的甲臜最终产品9,24。添加到细胞色素 C和琥珀酸钠基板内源性水平之间的控制和患病/突变体的组织 9正常化。过氧化氢 ​​酶增加一条，作为一项预防措施，以避免可能的污染从过氧化物酶的活性 9,22反应。 methosulfate吩嗪（PMS），中间电子载体，是叠氮化钠，呼吸链抑制剂，结合使用，以增加形成的最终反应产物 9,25 。尽管这一通知ATION，影响这个看似简单的检测结果的一些关键细节，除了特异性的控制和在技术的进步，都尚未提出。

Watch this Scientific Journal Video about Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry at JoVE.com

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

（COX / SDH），细胞色素C氧化酶/钠脱氢酶双标记方法允许直接可视化的新鲜冷冻组织切片中线粒体呼吸酶的缺陷。这是一个简单的组织化学技术，并在调查线粒体疾病，老化和衰老有关的疾病非常有用。

使用细胞色素线粒体呼吸功能的可视化 C氧化酶/琥珀酸脱氢酶（COX / SDH），双标组织化学

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

visualization-mitochondrial-respiratory-function-using-cytochrome-c

Research

JoVE Journal

Biology

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

36.5K 次观看.  Karolinska Institutet. （COX / SDH），细胞色素C氧化酶/钠脱氢酶双标记方法允许直接可视化的新鲜冷冻组织切片中线粒体呼吸酶的缺陷。这是一个简单的组织化学技术，并在调查线粒体疾病，老化和衰老有关的疾病非常有用。

视频: Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

科研

生物学

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

“体外骨骼肌微血管制备离体血管反应性的调查

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

评估肾细胞​​蛋白激酶C同工酶，线粒体功能及细胞活力的

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

颅面部发育中的SOX10可视化：使用枫转基因斑马鱼线时间推移共聚焦显微镜

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

在大规模的基因敲除<em&gt; C。线虫</em&gt;使用双链RNA料库产生失去功能的鲁棒表型

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

用奔放的整体体积描记测量呼吸功能的影响

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

高分辨率呼​​吸测量，以评估线粒体功能的通透和完整细胞

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

研究线粒体结构与功能<em&gt;果蝇</em&gt;卵巢

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.