在这个实验中，C2C12成肌细胞在海马细胞培养板的密度接种。建立基准利率基础耗氧量（OCR）和细胞外酸化（ECAR）率测量。 1。细胞注射这些细胞代谢由三个增加不同的化合物（继承），这种转变细胞的生物能个人资料的困扰。其中一组将作为控制，运行控制的“化合物”添加的媒体。 <ol><li>第一次注射寡。寡抑制ATP的合成，阻断质子通道 F O部分ATP合酶（综合五） 。在线粒体的研究，它是用来防止呼吸状态3（磷酸）。与细胞，它可以用来区分氧气消耗量的百分比，ATP的合成和需要克服自然的跨线粒体膜质子漏的氧气消耗量的百分比。 </li><li>第二次注射FCCP。 FCCP（羰基氰化物- P - trifluoromethoxyphenylhydrazone）是一个载体，是一个移动的离子载体。这是因为它扰乱运输氢离子穿过线粒体膜，而不是ATP合酶（综合五）的质子通道的ATP合成偶联代理。这种线粒体膜电位的崩溃导致的快速消耗能源和氧气，无ATP生成。在这种情况下，OCR和ECAR将增加，OCR由于偶联，ECAR细胞试图利用糖酵解产生ATP来维持自己的能量平衡。 FCCP治疗可用于计算的“备用”的定义是最大失控OCR和初步基础OCR之间的定量差异的细胞的呼吸能力。有人曾提出一些备用的呼吸能力，即使在最大的生理或病理生理刺激的条件下维护定义的活力和/或细胞的生存是一个主要因素。细胞的反应能力，能源需求增加的情况下强调的是影响了很大一部分的生物能量的线粒体能力。这种生物能的能力是由几个因素，包括能力的细胞线粒体和功能的电子传输所涉及的酶的能力提供基板，阻止开采</li><li>在第三次注射，鱼藤酮，复杂的I抑制剂，加入到细胞中。这将关闭线粒体呼吸，使线粒体和非线粒体的分数来计算的促进呼吸。人们将观察在OCR减少由于线粒体功能受损，在ECAR随之增加的细胞转移到更糖酵解的状态，以保持其能量平衡 鱼藤酮是一种线粒体抑制剂，以防止在复杂的Fe - S中心我泛醌（辅酶Q）的电子转移。我抑制这种复杂防止在NADH的潜在能量，在ATP的形式转换成可用能量。 </li></ol> 2。试剂和材料<ol><li>寡，FCCP，鱼藤酮的解决方案（海马美图压力测试套件） </li><li> DMEM运行Media（海马＃100965-000） </li><li> DMSO（Sigma公司D8418） </li><li>蒸馏水（Gibco公司15230-170） </li><li>校准缓冲液（海马生命科学） </li></ol> 3。生长介质<ol><li> 500毫升的DMEM（GIBCO 11965-092） </li><li> 10％胎牛血清（Hyclone公司SH90070.03） </li><li> 5毫升宾夕法尼亚/链球菌（GIBCO 15140-122） </li><li> 5毫升丙酮酸钠（Sigma公司S8636） </li><li> 5毫升Glutamax（GIBCO 35050-061） </li></ol> 4。播种协议<ol><li>的细胞接种于XF96细胞文化板10,000个细胞/孔 100μL培养液，并置于37℃ 与10％的CO 2 ° C培养箱中培养。 </li><li>在1小时内，细胞将坚持XF96细胞培养板。 </li><li> XF96 24小时后播种的检测细胞。 </li></ol> 5。检测模板的制备<ol><li>使用分析向导（附录I），产生一个与以下组的布局模板： <img src="/files/ftp_upload/2511/2511fig1.jpg" alt="图1" /> 图1井的网格布局，确定列和组分配</li></ol> 6。复方制剂<ol><li>准备下列化合物在XF的DMEM含量媒体如下： 10 UM寡，30.0 UM FCCP，20.0微米鱼藤酮， 这些浓度的化合物注射入井时将要进行的的10倍稀释。工作浓度： 1 UM寡，3.0 UM FCCP，2.0μM鱼藤酮</li></ol> 7。媒体的变化和细胞制备<oL&gt; <li>广场上的XF准备站的单元板</li><li>设置最终成交量中等，每孔160μL。 </li><li>在37℃培养箱中孵育60分钟，让细胞平衡与检测介质前没有的CO 2。 </li></ol> 8。载入传感器盒<ol><li>暖化合物为37 ° C装货前对传感器的墨盒装入喷油器的端口如下化合物： <ol><li>列1-4：负载16，18和20μLXF含量媒体（DMEM）到端口A，B和C，分别。 </li><li>列5-12： 加载到端口16μL寡一个 加载到端口B 18μLFCCP 装入端口彗星20μL鱼藤酮</li></ol></li></ol> 9。协议命令<table border="1"><tbody><tr><td> 命令 </td><td> 时间（min） </td><td> 港口 </td></tr><tr><td>校准</td><td></td><td></td></tr><tr><td>平衡</td><td></td><td></td></tr><tr><td>环路启动</td><td> 3X </td><td></td></tr><tr><td>组合</td><td> 3 </td><td></td></tr><tr><td>等待</td><td> 2 </td><td></td></tr><tr><td>测量</td><td> 3 </td><td></td></tr><tr><td>循环结束</td><td></td><td></td></tr><tr><td>注入</td><td></td><td>一</td></tr><tr><td>环路启动</td><td> 2X </td><td></td></tr><tr><td>组合</td><td> 3 </td><td></td></tr><tr><td>等待</td><td> 2 </td><td></td></tr><tr><td>测量</td><td> 3 </td><td></td></tr><tr><td>注入</td><td></td><td>乙</td></tr><tr><td>环路启动</td><td> 2X </td><td></td></tr><tr><td>组合</td><td> 3 </td><td></td></tr><tr><td>等待</td><td> 2 </td><td></td></tr><tr><td>测量</td><td> 3 </td><td></td></tr><tr><td>注入</td><td></td><td>彗星</td></tr><tr><td>环路启动</td><td> 2X </td><td></td></tr><tr><td>组合</td><td> 3 </td><td></td></tr><tr><td>等待</td><td> 2 </td><td></td></tr><tr><td>测量</td><td> 3 </td><td></td></tr><tr><td>完</td><td></td><td></td></tr></tbody></table> 表1。协议命令

<ol>
	<li>Choi, W. S., Gerencser, A. A., Nicholls, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioenergetic+analysis+of+isolated+cerebrocortical+nerve+terminals+on+a+microgram+scale:+spare+respiratory+capacity+and+stochastic+mitochondrial+failure.">Bioenergetic analysis of isolated cerebrocortical nerve terminals on a microgram scale: spare respiratory capacity and stochastic mitochondrial failure.</a> J Neurochem. 109, 1179-1191 (2009).</li><li>Hill, B. G., Dranka, B. P., Zou, L., Chatham, J. C., Darley-Usmar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+the+bioenergetic+reserve+capacity+in+response+to+cardiomyocyte+stress+induced+by+4-hydroxynonenal.">Importance of the bioenergetic reserve capacity in response to cardiomyocyte stress induced by 4-hydroxynonenal.</a> Biochem J. 424, 99-107 (2009).</li><li>Liu, J., Cao, L., Chen, J., Song, S., Lee, I. H., Quijano, C., Liu, H., Keyvanfar, K., Chen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmi1+regulates+mitochondrial+function+and+the+DNA+damage+response+pathway.">Bmi1 regulates mitochondrial function and the DNA damage response pathway.</a> Nature. , 459-7245 (2009).</li><li>Malmgren, S., Nicholls, D. G., Taneera, J., Bacos, K., Koeck, T., Tamaddon, A., Wibom, R., Groop, L., Ling, C., Mulder, H., Sharoyko, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+coupling+between+glucose+and+mitochondrial+metabolism+in+clonal+beta-cells+is+required+for+robust+insulin+secretion.">Tight coupling between glucose and mitochondrial metabolism in clonal beta-cells is required for robust insulin secretion.</a> J Biol Chem. 284, 32395-32404 (2009).</li><li>Dranka, B. P., Hill, B. G., Darley-Usmar, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+reserve+capacity+in+endothelial+cells:+The+impact+of+nitric+oxide+and+reactive+oxygen+species.">Mitochondrial reserve capacity in endothelial cells: The impact of nitric oxide and reactive oxygen species.</a> Free Radic Biol Med. 48, 905-914 (2010).</li><li>Perez, J., Hill, B. G., Benavides, G. A., Dranka, B. P., Darley-Usmar, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cellular+bioenergetics+in+smooth+muscle+cell+proliferation+induced+by+platelet-derived+growth+factor.">Role of cellular bioenergetics in smooth muscle cell proliferation induced by platelet-derived growth factor.</a> Biochem J. 428, 255-267 (2010).</li><li>Mor&#225;n, M., Rivera, H., S&#225;nchez-Arag&#243;, M., Bl&#225;zquez, A., Merinero, B., Ugalde, C., Arenas, J., Cuezva, J. M., Mart&#237;n, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+bioenergetics+and+dynamics+interplay+in+complex+I-deficient+fibroblasts.">Mitochondrial bioenergetics and dynamics interplay in complex I-deficient fibroblasts.</a> Biochim Biophys Acta. , 1802-185 (2010).</li><li>C&#225;rdenas, C., Miller, R. A., Smith, I., Bui, T., Molg&#243;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+regulation+of+cell+bioenergetics+by+constitutive+InsP3+receptor+Ca2++transfer+to+mitochondria.">Essential regulation of cell bioenergetics by constitutive InsP3 receptor Ca2+ transfer to mitochondria.</a> Cell. , 142-142 (2010).</li></ol>

吴敏，每博詹森，乔治W ·罗杰斯，和大卫A Ferrick海马生物科学，它产生在这篇文章中精选的试剂和仪器仪表的员工。

这个实验是来自经典的实验探测线粒体功能和作为一个建立更为复杂的实验，旨在了解，在细胞的新陈代谢，线粒体功能和整体生物能的各种变化的框架。 应在本实验中使用的所有化合物的浓度提供了极大的作用的优化。也就是说，必须执行单独的滴定实验，以确定这些值。这是与FCCP尤其重要，作为滴定曲线往往是相当尖锐的，太多FCCP实际上可以减少在OCR的回应。典型的范围（终浓度），以测试将是： <ul><li> 0.1 - 1.0微克/毫升的寡</li><li> 0.1 - 5.0 UM FCCP </li><li> 0.1 - 1.0 UM鱼藤酮</li></ul>请注意，每个上述化合物（尤其是FCCP）的反应将被检测媒体组成（基本类型，葡萄糖，丙酮酸，存在/ BSA的情况下，等）的影响。此外，如果XF检测媒体组成的改变，优化将需要重新执行。丙酮酸的存在和浓度是特别重要的，在取得最大呼吸能力由于FCCP。海马生物科学的观察，在一些细胞系，遗漏丙酮酸废除的细胞的能力，以最大限度地回应（基线以上）FCCP。通常情况下，浓度为1-10毫米丙酮酸，应进行测试，以了解最佳浓度的丙酮酸，以获得最大的呼吸。请注意，[丙酮酸]和[糖]，可能需要“跨滴定”，以获得最佳的媒体实验条件。 OCR随时间变化和另一个显示ECAR随时间的图形显示，本次实验的典型结果介绍如下： <img src="/files/ftp_upload/2511/2511fig2.jpg" alt="图2" /> 图2。OCR的随时间变化<img src="/files/ftp_upload/2511/2511fig3.jpg" alt="图3" /> 图3。ECAR随时间变化在这里，我们观察到OCR和ECAR预期反应的细胞与每个连续的复合处理。对于寡，OCR作为ATP合成阻断线粒体复合物V.由于细胞不能合成ATP通过OXPHOS，他们恢复糖酵解，以满足他们对ATP的需求下降，因此，我们观察ECAR增加。如前所示，FCCP行为作为偶联剂。由于细胞必须克服跨线粒体膜的质子漏，OCR显着增加，更多的氧气被消耗掉多余的质子穿过线粒体膜泵。最后，鱼藤酮抑制线粒体复合物I和综合第三，分别，这会导致停止在电子传递链的电子流，从而氧气消耗量大幅降低。 <img src="/files/ftp_upload/2511/2511fig4.jpg" alt="图4" /> 图4呼吸参数。 除了呼吸和ECAR的预期变化，呼吸参数，可从这个数据。这是总结在上图中： 这里我们可以看到，我们可能会获得有关的基底细胞呼吸，致力于ATP的生产以及致力于维持质子梯度（H +泄漏）的金额的氧气消耗量的百分之的信息。此外，我们可能会耦合的呼吸（有时也被称为呼吸能力作为备用）的条件下获得最大的呼吸频率，最后，我们可以判断氧气消费量不因线粒体进程。 一个研究数量迅速增加是采用这种线粒体的个人资料，评估细胞的生物能，确定线粒体功能障碍和预测细胞的能力，以应对压力和/或侮辱。欲了解更多的信息和对本实验方法和备用呼吸能力的想法的细节， 请参阅参考1-8以下出版物。

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<td>Oligomycin, FCCP, Rotenone and Antimycin A Solutions</td>
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<td>D8418</td>
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bioenergetic profile experiment using c2c12 myoblast cells

的能力，测量细胞的新陈代谢和理解线粒体功能障碍，使全世界的科学家了解肥胖的线粒体功能的作用，糖尿病，衰老，癌症，心血管功能和安全性的毒性，以推动他们的研究。 细胞代谢底物的吸收，如氧，葡萄糖，脂肪酸，谷氨酰胺，并随后通过一系列酶控制的氧化和还原反应的能量转换过程中。在生产ATP，释放的热量和化学副产品，如乳酸和CO 2到细胞外环境，这些细胞内的生化反应结果。 进入细胞的生理状态，并改变这些细胞的状态可以得到宝贵的见解，通过测量细胞消耗氧气，线粒体呼吸的指标利率 - 耗氧率 - 或OCR。细胞也通过糖酵解生成ATP，即：葡萄糖转化为乳酸，独立的氧气。在培养的井，乳酸是质子的主要来源。测量乳酸产生通过间接释放到细胞外的周边中等质子，从而导致介质酸化提供了细胞外酸化率 - 或ECAR。 在这个实验中，C2C12成肌细胞在海马细胞培养板的密度接种。建立基准利率基础耗氧量（OCR）和细胞外酸化（ECAR）率测量。细胞代谢扰动由三个增加不同的化合物（继承），这种转变细胞的生物能个人资料。 这个实验是来自一个经典的实验，以评估线粒体作为一个框架，建立在更为复杂的实验，旨在了解双方的生理和病理生理线粒体功能和预测细胞的能力，以应对压力和/或侮辱。

Watch this Scientific Journal Video about Bioenergetic Profile Experiment using C2C12 Myoblast Cells at JoVE.com

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

在细胞线粒体功能分析的方法的描述。线粒体产生的个人资料提供线粒体的功能，可以在一次实验中测量的四个参数：基础呼吸速率，ATP挂钩呼吸，质子漏，和储备能力。

使用C2C12成肌细胞的生物能简介实验

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

bioenergetic-profile-experiment-using-c2c12-myoblast-cells

Research

JoVE Journal

Biology

50.2K 次观看.  Novato, CA. 在细胞线粒体功能分析的方法的描述。线粒体产生的个人资料提供线粒体的功能，可以在一次实验中测量的四个参数：基础呼吸速率，ATP挂钩呼吸，质子漏，和储备能力。

视频: Bioenergetic Profile Experiment using C2C12 Myoblast Cells

Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well know to have insecticidal properties. One response of tobacco plants to herbivory, or being gnawed upon by insects, is to increase the production of this neurotoxic alkaloid. Here, we will demonstrate how to set up an experiment to address this question of whether a tobacco-adapted strain of the green peach aphid, Myzus persicae, can tolerate higher levels of nicotine than the a strain of this insect that does not infest tobacco in the field.

In this video and assay is demonstrated that tests the tolerance of nicotine to two types of aphids one that infests tobacco plants in the field and one that does not.

使用人工饲料实验测试蚜虫的尼古丁公差

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well ...

科研

生物学

Morris Water Maze Experiment

The Morris water maze is widely used to study spatial memory and learning. Animals are placed in a pool of water that is colored opaque with powdered non-fat milk or non-toxic tempera paint, where they must swim to a hidden escape platform. Because they are in opaque water, the animals cannot see the platform, and cannot rely on scent to find the escape route. Instead, they must rely on external/extra-maze cues. As the animals become more familiar with the task, they are able to find the platform more quickly. Developed by Richard G. Morris in 1984, this paradigm has become one of the "gold standards" of behavioral neuroscience.

The Morris water maze is a well-accepted tool used to document the involvement of the hippocampus in a behavioral task.

Morris水迷宫实验

The Morris water maze is widely used to study spatial memory and learning. Animals are placed in a pool of water that is colored opaque with powdered ...

Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells.  A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results.  This procedure is readily adaptable to mammalian cells.

This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.

真核Polyribosome剖面分析

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

斑马鱼胚胎的代谢谱分析

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

原发性生肌前体细胞/成肌细胞从基底哺乳动物支系文化准备

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

代谢谱的实时分析<em&gt;离体</em&gt;鼠标肠隐窝类器官培养

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

胞嘧啶修饰形式使用敏感免疫组化检测

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Exploiting Live Imaging to Track Nuclei During Myoblast Differentiation and Fusion

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear positioning occurs during skeletal muscle differentiation. Muscle fibers (myofibers) are multinucleated cells formed by the fusion of muscle precursor cells (myoblasts) derived from muscle stem cells (satellite cells) that undergo proliferation and differentiation. Correct nuclear positioning within myofibers is required for the proper muscle regeneration and function. The common procedure to assess myoblast differentiation and myofiber formation relies on fixed cells analyzed by immunofluorescence, which impedes the study of nuclear movement and cell behavior over time. Here, we describe a method for the analysis of myoblast differentiation and myofiber formation by live cell imaging. We provide a software for automated nuclear tracking to obtain a high-throughput quantitative characterization of nuclear dynamics and myoblast behavior (i.e., the trajectory) during differentiation and fusion.

Skeletal muscle differentiation is a highly dynamic process, which particularly relies on nuclear positioning. Here, we describe a method to track nuclei movements by live cell imaging during myoblast differentiation and myotube formation and to perform a quantitative characterization of nuclei dynamics by extracting information from automatic tracking.

在成细胞分化和融合过程中利用活成像跟踪核

Nuclear positioning within cells is important for multiple cellular processes in development and regeneration. The most intriguing example of nuclear ...