我们感谢作者介绍的线粒体生物学和许多有益的见解和意见，在此过程中领域的教授A.维诺格拉多夫和维拉Grivennikova博士。作者感谢夫人阿曼达McKintock从皇后大学媒体与视频准备的帮助办公室。

简介从大鼠心脏线粒体是细胞代谢的过去和目前的研究中最常见的准备工作之一。它们可以快速，可靠地获得了很大的数量从野生型基因敲除动物。一般程序包括胰蛋白酶，分馏和差速离心组织消化。 有几个条件，要保持在线粒体的分离，从各种组织，包括心脏（图1）。 <ul><li>该组织已被彻底剁碎，因为它直接影响产量获得线粒体。心脏组织是最好讳言小弯剪，可以接着使用Latapie组织磨床，或者，如果一个组织研磨机无法使用，与插座孔直径约1.0毫米直径的压蒜。 </li><li>这是必要的温度维持在每一步的解决方案。因此整个过程必须在寒冷的房间和所有仪器和解决方案已提前准备和冷却。如果没有保存的温度条件，稳定的几个属性的准备可能会丢失。线粒体的结构是非常敏感的冻结，以便overfreezing暂停（例如在离心）是不允许的，尤其是如果研究是重要的主动运输。 </li><li>一个重要的参数是该过程的时间长度;隔离应尽快进行，并获得准备立即使用。因此，手术器械，解决方案和设备要提前做好准备。 </li></ul>材料与仪器仪器<ol><li>直剪刀，尖</li><li>弯剪刀</li><li>镊子</li><li>培养皿</li><li>小漏斗+尼龙过滤片（预洗） </li><li> 6烧杯50毫升</li><li>磁力搅拌器和两个冰浴</li><li>一个大冰浴</li><li> 2个小型的磁力棒</li><li>为颗粒铲刮</li><li>离心管</li><li> Latapie组织记者（可取代压蒜） </li><li>铁氟龙玻璃均质20毫升和12毫升</li><li>废物烧杯</li><li>最终线粒体悬浮和测定蛋白质的样品的Eppendorf管</li><li>冰浴</li></ol>解决方案（每2只计算）： <ol><li>洗涤缓冲液：0.3米蔗糖，10毫米的HEPES（pH值= 7.2），0.2毫米EDTA（1000毫升） </li><li>分离缓冲液：蔗糖0.3米，10毫米的HEPES（pH = 7.4的），0.2毫米EDTA，1 mg / ml的BSA（Sigma公司A6003）（100毫升洗液编制）。牛血清白蛋白可以取代更便宜的脂肪酸类似物。相同的缓冲区或等渗的变化，可以用来作为Resuspending缓冲。 </li><li>胰蛋白酶（Sigma公司T8003）解决方案：在1MM盐酸2.5mg/ml（0.5毫升） </li><li>从大豆的胰蛋白酶抑制剂（Sigma公司T9128）6.5 mg/10毫升隔离缓冲</li></ol>协议上图所示的程序的总体方案。 1。心应冷却和洗涤缓冲液，心室组织切除，切碎，并在光解治疗胰蛋白酶均质洗涤。悬挂在低速离心和更高的速度再次获得上清离心收集线粒体。根据计划实验resuspending缓冲区可取代任何其他的等渗溶液。 动物，终止与英国“动物（科学规程）法”的规定。“颈椎脱位由斩首。提取后，立即斩首的动物心脏，这是必要的，因此，没有任何动物终止和心脏提取之间的延迟。如果平行隔离肝线粒体预计应大鼠实验前禁食过夜。 <ol><li>准备三个含有40毫升的洗涤缓冲液的烧杯。在3至5分钟，然后再继续下一步的冰盐浴冷却他们。确保不overfreeze冰晶云后开始出现，并立即使用。 </li><li>断头处死大鼠，打开胸腔，并提取心脏。小切口，然后立即转移的心冰冷的解决方案中的第一个烧杯。挤压心脏，以清除血液，并放入第二个烧杯。重复此操作，每个心。 </li><li>干滤纸上的心。卸下所有的脂肪，血块，耳廓和筋膜和池心室组织。 </li><li>百果汇集的组织，直到5分钟左右的小弯剪刀在培养皿上冰粒子的大小约1-2毫米。 </li><li>组织按放入剁碎的组织和传递它通过。请注意，大量的材料可以在按下继续这样做从记者的墙壁和底部收集所有的心脏组织。 </li><li>转移到烧杯中的物质与50毫升的洗涤缓冲液和搅拌。通过尼龙过滤器，然后过滤暂停。洗液洗净剁碎组织在过滤器上的3倍，并丢弃滤液。 </li><li>转移到20毫升的洗涤缓冲液洗涤组织和放置在冰水浴磁力搅拌器。加入0.5毫升的胰蛋白酶不断搅拌下的解决方案，并启动计时器。准备另一磁力搅拌器，冰浴和第二个50毫升的烧杯。 </li><li>在第 4分钟，简单地通过使用几个和向下招松散安装一个小玻璃均质聚四氟乙烯（10 - 15毫升），以驱散暂停部分均质暂停部分。均质后转移到第二个烧杯中暂停。 第 9分钟的重复过程，所以，你总执行暂停均质两次。孵育15分钟后，添加10毫升的隔离缓冲含有胰蛋白酶抑制剂和孵育1分钟。 </li><li>暂停，再通过过滤器的尼龙过滤器，并保存滤液。收集的微粒分数左一个过滤器，均质，洗涤缓冲液15-20毫升1分钟。 </li><li>合并滤液，离心15分钟在600克匀浆。 </li><li>仔细筛选到一个新的离心管中产生的上清液。避免污染颗粒，离心15分钟8500克再次。 </li><li>高速离心后，弃上清，用1毫升的缓冲区丢弃的蓬松的白色外缘层冲洗沉淀2-3次。分手的颗粒，但要避免用锅铲在底部红细胞的红点。如果血细胞得到洛斯，删除从均质前悬挂红色位。 </li><li>用小均质或交替轻柔吹打重悬沉淀。更多Isolationbuffer悬浮液稀释，离心15分钟8500克再次。 </li><li>弃上清，重悬在Resuspending缓冲区（选择的等渗缓冲）沉淀，离心一次。 </li><li>由此产生的棕色颗粒包含洗完整的线粒体。在您所选择的等渗缓冲的体积非常小的重悬沉淀。 </li></ol><img src="/files/ftp_upload/2202/2202fig1.jpg" alt="图1" /> 图1展示的隔离大鼠心脏线粒体的程序一步一步的计划。顶端部分，1-9阶段：组织受阻，胰蛋白酶处理和均质化;底部的一部分，阶段10-15：差速离心。

<ol>
	<li>Jacobus, W. E., Saks, V. A. <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+heart+mitochondria:+changes+in+its+kinetic+properties+induced+by+coupling+to+oxidative+phosphorylation.">Creatine kinase of heart mitochondria: changes in its kinetic properties induced by coupling to oxidative phosphorylation.</a> Arch. Biochem. Biophys. 219, 167-178 (1982).</li><li>Gostimskaya, I. S., Grivennikova, V. G., Zharova, T. V., Bakeeva, L. E., Vinogradov, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+assay+of+the+intramitochondrial+enzymes:+use+of+alamethicin+for+permeabilization+of+mitochondria.">In situ assay of the intramitochondrial enzymes: use of alamethicin for permeabilization of mitochondria.</a> Anal. Biochem. 313, 46-52 (2003).</li></ol>

迅速隔离与耦合的高度完整的线粒体协议详细描述。准备获得可用于功能或结构，对细胞的生物能学，蛋白质组学研究或线粒体DNA的分析和脂肪含量的研究。也可以进行主动运输的离子和代谢中间体的生物物理研究。这是可能的进一步过程中线粒体的准备，以隔离submitochondrial颗粒，外膜或氧化磷酸化的净化单独的组件。所描述的方法是修改的标准协议隔离雅各和Saks 1。准备线粒体的预期收益率大约为10-15毫克每心脏线粒体蛋白质和苹果酸/谷氨酸等准备不同的呼吸控制率8至12国IV周围O 2的0.12μmolxmin -1 XMG蛋白呼吸-1 23 ° C中的介质，包括0.25 M蔗糖氯化钾，10毫米，0.2毫米EDTA，5 mM磷酸钾（pH值8.0）与150微米ADP启动国家第三呼吸（确切的实验条件和补充，见文献2）。 应特别注意隔离程序开始前的几个技术细节。它是使用干净的聚碳酸酯或聚丙烯离心管的关键，因此应彻底刷干净的热水，然而，洗涤剂治疗应保持在最低，洗涤管。此外，它是更好地收集一切必要的玻璃器皿和仪器在寒冷的房间隔离的前一天。 在隔离烧杯烧瓶传输缓冲区时，а特别注意应的解决方案，防止滴冰水。处理组织时，应该强调的，因为即使短期斩首的动物和冷却的组织之间的时间延迟将导致部分耦合线粒体进行了心脏提取应尽快。快速冷却，除去心中的彻底清洗是必须获得标准的编制，血红蛋白及红细胞NADase。 胰蛋白酶是降解结缔组织蛋白组织的易感性增加在均质剪切力。应避免长时间暴露在蛋白酶组织，因为它在质量低劣的线粒体结果。 经过高速离心，离心管的墙壁应该用纸巾擦拭，删除任何墙壁上积累的脂肪物质的存款。 对于最终的再悬浮，它是更好地使用，以低量的最终缓冲区，以尽量减少在储存过程中（150至200μL心每缓冲区）线粒体功能的丧失。运输二价阳离子的研究没有螯合剂必须在最后的准备，因此线粒体应清洗数次与EGTA无介质隔离后的第一个小时内使用。

preparation of highly coupled rat heart mitochondria

在氧化磷酸化的的过程中在细胞内ATP生成的线粒体的功能，是举世公认的。在过去几十年中已经出现了重大进展，我们比其他能源发电的线粒体的功能的认识。这些措施包括在细胞凋亡中的作用，作为信号的细胞器，哺乳动物发育和老化细胞的新陈代谢和细胞增殖之间的协调所作出的贡献以及的。我们的生物过程由线粒体调制的理解是基于强劲隔离和处理实验动物组织的完整线粒体方法。从大鼠心脏线粒体是细胞代谢的过去和目前的研究，包括对敲除动物的研究中最常见的准备工作之一。 在这里，我们描述了一个详细的隔离与耦合的高度完整的线粒体快速的方法。大鼠心脏线粒体的这些准备工作是出色的功能和结构研究，对细胞的生物能，生物分子，蛋白质组研究和分析线粒体DNA，蛋白质和脂质的运输对象。

Watch this Scientific Journal Video about Preparation of Highly Coupled Rat Heart Mitochondria at JoVE.com

Preparation of Highly Coupled Rat Heart Mitochondria

我们描述а纯，高度耦合的大鼠心脏细胞生物能，生物物理测量学，蛋白质组学或线粒体DNA和脂质分析的功能或结构的研究线粒体隔离协议。

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 ...

preparation-of-highly-coupled-rat-heart-mitochondria

Research

JoVE Journal

Biology

22.7K 次观看.  University of Manchester. 我们描述а纯，高度耦合的大鼠心脏细胞生物能，生物物理测量学，蛋白质组学或线粒体DNA和脂质分析的功能或结构的研究线粒体隔离协议。

视频: Preparation of Highly Coupled Rat Heart Mitochondria

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

大鼠心脏中植入的工程组织

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

科研

生物学

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

成年大鼠心脏心肌细胞的原代培养

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

从酵母细胞线粒体的纯化

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

制备鼠尾肌腱的生物力学和Mechanobiological研究

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...

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 ...

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 ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

大鼠血清适用于哺乳动物全胚胎培养的制备

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

再加上高灵敏度指纹碳水化合物自动分析模块化高通量筛选胞外多糖平台

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

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 ...