我们感谢Fabiana Benatti她在视频的友好合作。我们也感谢支持我们的研究在这方面FAPESP（＃2007/51228-0）和CNPq（＃300133/2008-1）。

介绍为维持体力所必需的能量来自两个代谢来源：有氧运动和无​​氧代谢。虽然有氧代谢超过无氧代谢的效率（即，它产生的每摩尔ATP的更高的基板数量），通过无氧代谢产生的能量可以在很短的时间内提供的能量高的金额。任何情况下，需要极快的动作，这可能是决定性的。 每项运动都有运动技能，赋予独特的生理和代谢为特定的体育需求方面的具体特点。的代谢需求的最重要的方面是能源系统的活动所需的总能量的相对贡献。每个运动的具体需求来确定的是优化训练模式，营养战略和增补剂，可最大限度地提高发展的关键thletic性能。 有些运动是比较容易被复制在实验室环境中，从而有可能创建一个受控的环境中，运动员可以评估。这是例如，跑步和骑自行车的案件。可预见的变动构成这些运动，因此，它们很容易进行研究。使用一些简单的设备，它是可以模仿，运动员在训练和比赛的实际情况，如，执行同样的动作相当准确。事实上，这些运动已被更广泛地研究运动的科学家，一个更加完整和可靠的科学文献中受益。 另一方面，一些体育更难以在实验室中复制。这些运动是不可预测的，依赖于合作伙伴（S）和对手（S）的行动。这将导致无法在实验室中的竞争条件和ASSE无法准确地再现SS这些运动员无论是训练还是比赛期间，在该领域。也许是因为这些问题，他们已经收到从科学家的关注就少得多。这是大多数团队运动和许多单项体育1。 考虑到这些方面，我们的目的是描述如何评估能源系统在受控的实验室条件下是难以重现的体育差贡献。因为柔道是一个非常复杂和不可预测的运动，我们将使用一个例子作为柔道。然而，这里显示的概念，可适应不同的运动。 1。在休息的生理测量<ol><li>运动员的体重测量之前，他/她发起行使。 </li><li>在开始锻炼之前，收集从耳垂或指尖一个小休息的血液样本，并保持它在冰上，直到完成整个实验过程。 </li><li>之后，将CAL在最方便的位置，这取决于对运动的运动员将执行，并纪录五分钟的休息或基线耗氧量ibrated便携式气体分析仪。计量基准期间，运动员留在安静的站在他/她的脚（如果这次演习将在立姿），或坐在设备将用于（如果演习将在cycloergometer或执行任何类似的设备）。 </li></ol> 2。运动过程中的生理测量<ol><li>收集休息的血液样本和休息耗氧量后，你可能会问运动员开始，你正在学习的具体行使。便携式气体分析仪被放置在一个位置，不会干扰演习，演习不会损坏设备。整个演习期间，继续测量耗氧量。 </li></ol> 3。运动后生理测量</P&gt; <ol><li>运动耗氧量数据收集后，前10分钟关闭设备记录耗氧量。如果一个以上的运动员正在评估在同一天，总是校准气体分析仪。 </li><li>以确定运动后血乳酸峰值，收集小的血液样本后立即运动，三，五，七分钟后行使。继续，直到分析冰。 </li></ol> 4。血液样品处理和山顶血浆乳酸测定<ol><li>所有的血液样本，必须放置在含有类似的2％氟化钠溶液体积（即，如果你正在收集25μL血液，将在25μL2％氟化钠）的微管。 </li><li>当数据采集完成后，从纺纱2000Ğ样本为5分钟，在4°C的红细胞分离血浆</li><li>血浆乳酸，可确定通过各种的methods2 3。在我们的实验室中，我们使用电化学冰毒OD与一个automatized乳酸分析仪（黄河泉1500体育，俄亥俄州）的援助。 </li></ol> 5。计算<ol><li>计算净有氧代谢产生的能量减去其余耗氧量运动耗氧量。休息时消耗氧气通过乘以基线耗氧量的最后30秒的平均总运动持续时间。然后，使用梯形法计算运动耗氧量曲线下的面积。最后，运动耗氧量减去休息的耗氧量。 </li><li>的的厌氧alactic代谢（即ATP-CP的途径）的贡献是不能被视为过量运动后耗氧量4-6快速组件， 如图1所示。计算拟合产生由alactic系统的能量运动后耗氧量双向1 monoexponenti的动力学人曲线。这可以用数学软件（如Microcal公司原产版本7.0）的援助。基于模型最适合您的数据集（即，最低的残留物）的单或双指数曲线的选择。然后，使用的拟合方程（ 公式1）提供的条款，，计算alactic贡献，根据公式（2）。 <img src="/files/ftp_upload/3413/3413fig1.jpg" alt="fiugre 1" /> 图1一个典型的耗氧量，在休息期间和运动后，得到的曲线示意图插图。 公式1： <img src="/files/ftp_upload/3413/3413eq1.jpg" alt="式（1）" /> 公式（2）： <img src="/files/ftp_upload/3413/3413eq2.jpg" alt="公式（2）" /> 其中V O2（T）是在时间t的吸收氧气，五O2baseline摄氧量在基线，A是振幅，δ是延迟时间，τ是时间常数，1和2表示的快与慢的组件，分别。 </li><li>计算乳酸菌的厌氧系统的贡献，这是假设1毫米以上的乳酸休息值对应3毫升每公斤mass7身体消耗的氧气。因此，计算三角洲高峰血浆乳酸（即峰值血浆乳酸减去休息血浆乳酸）和乘以3，运动员的体重。毫升的氧气值，然后转换为L和能量（千焦），假设每个O 2的 1升等于20.92千焦耳。 </li><li>最后，各能源系统得到的结果总结，所以你必须在活动期间的总能量消耗，可以计算出每个系统的相对贡献。 </li></ol> 6。代表结果 图2描述了曲线运动期间和运动后耗氧量的代表在休息。在例如用在这里，运动员进行了五分钟的三个不同的柔道技术（O-宇智木薯粉，harai，五志与seoi-nage）（1抛出每15秒）8。这是一个典型的间歇性运动的反应。经过计算，我们得到了在柔道练习的贡献（ 见表1）能源系统的最终结果。 其他代表性的结果显示在表2。在这个例子中，不同竞争力的水平（即娱乐与精英）的室内攀岩在一个低难度的攀登路线进行了评估。为精英运动员和一个休闲运动员个人的结果显示（ 见表2）。 <table border="1"><tbody><tr><td rowspan="2"></td><td colspan="2"> seoi-nague </td><td colspan="2"> harai，五志 </td><td colspan="2"> O型宇智-加里 </td></tr><tr><td> 千焦 </td><td> ％ </td><td> 千焦 </td><td> ％ </td><td> 千焦 </td><td> ％ </td></tr><tr><td>厌氧alactic </td><td> 46±20 </td><td> 16.3±2.8 </td><td> 43±21 </td><td> 16.1±2.7 </td><td> 36±22 </td><td> 14.6±2.8 </td></tr><tr><td>有氧</td><td> 223±66 </td><td> 82.2±2.9 </td><td> 211±66 </td><td> 82.3±3.8 </td><td> 196±74 </td><td> 84.0±3.8 </td></tr><tr><td>无氧乳酸</td><td> 4±2 </td><td> 1.5±0.7 </td><td> 5±5 </td><td> 1.6±1.4 </td><td> 4±4 </td><td> 1.5±1.1 </td></tr><tr><td> 总 </td><td> 273±86 </td><td>- </td><td> 259±91 </td><td> - </td><td> 237±99 </td><td> - </td></tr><tr><td> 总计（千焦耳/分钟） </td><td> 51.9±8.7 </td><td> - </td><td> 49.4±8.9 </td><td> - </td><td> 45.3±19.6 </td><td> - </td></tr></tbody></table> 表1。代表的总能量消耗和能源系统的贡献，在三个不同的柔道练习的结果。 <table border="1"><tbody><tr><td> 竞技水平 </td><td> 健美操（％） </td><td> 无氧乳酸（％） </td><td> 的厌氧Alactic（％） </td><td> 总计（千焦） </td><td> 总计（千焦耳/秒） </td></tr&gt; <tr><td>精英</td><td> 40 </td><td> 8 </td><td> 52 </td><td> 70.4 </td><td> 1.00 </td></tr><tr><td>娱乐</td><td> 40 </td><td> 15 </td><td> 45 </td><td> 96.1 </td><td> 1.15 </td></tr></tbody></table> 表2。代表个人数据的总能量消耗和能源系统的贡献，在一个低难度的攀登路线。 <img src="/files/ftp_upload/3413/3413fig2.jpg" alt="图2" /> 图2。代表结果在5分钟柔道运动。

<ol>
	<li>Franchini, E., Del Vecchio, F. B., Matsushigue, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+profiles+of+elite+judo+athletes.">Physiological profiles of elite judo athletes.</a> Sports Med. 41, 147-166 (2011).</li><li>Bergmeyer, H. U., Bergmeyer, J., Grassl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods of enzymatic analysis. , (1983).</li><li>Passonneau, J. V., Lowry, O. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+Analysis.">Enzymatic Analysis.</a> A Practical Guide. , (1993).</li><li>Beneke, R., Pollmann, C., Bleif, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+anaerobic+is+the+Wingate+Anaerobic+Test+for+humans.">How anaerobic is the Wingate Anaerobic Test for humans.</a> Eur. J. Appl. Physiol. 87, 388-392 (2002).</li><li>Beneke, R., Beyer, T., Jachner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energetics+of+karate+kumite.">Energetics of karate kumite.</a> Eur. J. Appl. Physiol. 92, 518-523 (2004).</li><li>Bertuzzi, R. C. D., Franchini, E., Kokubun, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+system+contributions+in+indoor+rock+climbing.">Energy system contributions in indoor rock climbing.</a> Eur. J. Appl. Physiol. 101, 293-300 (2007).</li><li>di Prampero, P. E., Ferretti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+energetics+of+anaerobic+muscle+metabolism:+a+reappraisal+of+older+and+recent+concepts.">The energetics of anaerobic muscle metabolism: a reappraisal of older and recent concepts.</a> Respir. Physiol. 118, 103-115 (1999).</li><li>Franchini, E., Bertuzzi, R. C. D., Degaki, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+Expenditure+in+Different+Judo+Throwing+Techniques.+Proceedings+of+first+joint+international+pre-Olympic+conference+of+sports+science+and+sports+engineering,+vol+II.">Energy Expenditure in Different Judo Throwing Techniques. Proceedings of first joint international pre-Olympic conference of sports science and sports engineering, vol II.</a> Bio-mechanics and sports engineering. , 55-60 (2008).</li><li>Calmet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+ecological+research+in+judo.">Developing ecological research in judo.</a> Percept. Mot. Skills. 105, 646-648 (2007).</li><li>Mello, F. D., Bertuzzi, R. C., Grangeiro, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+systems+contributions+in+2,000+m+race+simulation:+a+comparison+among+rowing+ergometers+and+water.">Energy systems contributions in 2,000 m race simulation: a comparison among rowing ergometers and water.</a> Eur. J. Appl. Physiol. 105, 615-619 (2009).</li><li>Bertuzzi, R. C., Franchini, E., Ugrinowitsch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+MAOD+using+only+a+supramaximal+exhaustive+test.">Predicting MAOD using only a supramaximal exhaustive test.</a> Int. J. Sports Med. 31, 477-481 (2010).</li></ol>

作者宣称，他们有没有关于这项研究的利益冲突。

我们已经显示了野兔的方法，可用于连续和间歇性运动。该方法的最大优点是它可以适应，是很难被模仿在受控的实验室设置的锻炼和体育。此外，这是唯一可用的方法，能够区分三种不同的能源系统的贡献。因此，该方法允许运动的研究，具有很大的相似性，以实际情况，提供理想的生态效度的研究9。例如，德梅洛等人最近研究表明，在2000米水赛艇比赛的的糖酵解贡献的只有7％，这意味着，赛艇的性能主要取决于有氧代谢。同样，贝内克等人的研究证实，在能源的主要来源之一，最常用的厌氧测试，Wingate无氧试验，是无氧代谢（20％有氧; 30％ALACTIC和糖酵解50％）。由本集团最近的研究也具有室内攀岩和柔道8日报道，在这个例子中的能量贡献。事实上，充满活力的贡献知识，增进机能的策略，培训机构的发展是至关重要的，甚至验证测试。 这种方法有一定的局限性。首先，设备的成本是有点高，需要专门训练的人员。其次，可以用这种技术模仿虽然大多数的体育运动，它是没有任何运动的类型，可使用便携式气体分析仪研究。最后，血浆乳酸并不完全代表总骨骼肌乳酸生产活动时，用这种方法得到的结果可以被视为一个新陈代谢的需求推估在演习过程中，精确量化，而不是一个充满活力的贡献。然而，这是我只验证thod可用11能够区分三种不同的能源系统的贡献。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>评论</th></tr><tr><td> YSI 1500体育</td><td>黄河泉</td><td>该设备允许一个快速简便的血浆乳酸测定</td></tr><tr><td> K4的B2 </td><td>康是美</td><td>此设备是必不可少的测量耗氧量，整个演习</td></tr><tr><td>软件Microcal公司6.0 </td><td>起源</td><td>此软件（或任何其他具有类似功能）将是有益的计算</td></tr></tbody></table>

determining the contribution of the energy systems during exercise

代谢需求的最重要方面之一，是一个给定的体力活动所需的总能量的能源系统的相对贡献。虽然有些运动是比较容易被复制在实验室（例如，跑步和骑车），体育是在受控的情况下进行复制和研究更加困难。此方法介绍了如何在受控的实验室条件下是难以模仿的运动中的能量系统的评估差的贡献。这里显示的概念，可以适应几乎任何运动。 以下的生理变量，将所需：休息耗氧量，运动耗氧量，运动后耗氧量，其余的血乳酸浓度和运动后血浆峰乳酸。计算的有氧代谢的贡献，您将需要在休息和演习中的氧气消耗。通过使用梯形法，耗氧量曲线下面积计算运动过程中，其余耗氧量减去相应面积。计算的alactic无氧代谢的贡献，运动后耗氧量曲线进行调整，以单声道或双指数模型（选择一个最适合）。然后，用拟合方程的条款计算厌氧alactic的代谢，如下：ATP-CP的代谢= 1（毫升S -1）XT（S）。最后，计算乳酸菌的厌氧系统的贡献，乘以3，运动员的体重（ML中的结果，然后转换为L到千焦）血浆乳酸峰值。 该方法可用于连续和间歇性运动。这是一个非常有趣的方法，因为它可以适应锻炼和运动，在受控环境中难以被模仿。此外，这是唯一一个vailable方法能够区分三种不同的能源系统的贡献。因此，该方法允许运动的研究，具有很大的相似性，以实际情况，提供理想的生态效度研究。

Watch this Scientific Journal Video about Determining the Contribution of the Energy Systems During Exercise at JoVE.com

Determining the Contribution of the Energy Systems During Exercise

该协议允许专注于运动和体育科学的研究，以确定期间各种大型演习的总能量消耗的三种不同的能源系统的相对贡献。

确定的能源系统，在运动的贡献

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given ...

determining-the-contribution-of-the-energy-systems-during-exercise

Research

JoVE Journal

Biology

41.1K 次观看.  University of Sao Paulo. 该协议允许专注于运动和体育科学的研究，以确定期间各种大型演习的总能量消耗的三种不同的能源系统的相对贡献。

视频: Determining the Contribution of the Energy Systems During Exercise

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

计数和确定培养细胞的可行性

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

科研

生物学

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

小鼠乳腺上皮细胞形成过程中Lactogenic分化的微球体

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

肚子里的龙虾总夹层， Homarus Americanus</em

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.
Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2.
To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1.
The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

The Scepter Cell Counter is a handheld automated device that can be used to count cells, monitor cell diameter and volume, and be used to check the health and quality of cellular populations from one culture to the next.

确定在细胞培养的细胞数量，使用权杖细胞计数

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

卵裂球外植体测试胚胎发育过程中细胞命运的承诺

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

恒化器中微生物系统生物学中的应用

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

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 ...

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa have the potential to impair animal welfare and research. The fish are typically analyzed post mortem to detect microbes. The use of sentinels is a suggested way to improve the sensitivity of the surveillance and to reduce the number of animals to sample. The setting of a pre-filtration sentinel tank out of a recirculating system is described. The technique is developed to prevent water pollution and to represent the fish population by a careful selection of age, gender, and strains. In order to use the minimum number of animals, techniques to screen the environment are also detailed. Polymerase Chain Reaction (PCR) on surface sump swabs is used to significantly improve the detection of some prevalent and pathogenic mycobacterial species such as Mycobacterium fortuitum, Mycobacterium haemophilum, and Mycobacterium chelonae. Another environmental method consists of processing the sludge at the bottom of a holding tank or sump to look for P. tomentosa eggs. This is a cheap and fast technique that can be applied in quarantine where a breeding device is submerged into the holding tank of imported animals. Finally, PCR is applied to the sludge sample and A. hydrophila is detected at the sump&#39;s bottom and surface. Generally, these environmental screening techniques applied to these specific pathogens have led to an increased sensitivity compared to the testing of pre-filtration sentinels.

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

This protocol describes the use of sump swabs and sludge analysis of zebrafish systems, which leads to increased detection compared to the sole use of sentinels to detect pathogens such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa. A system to monitor P. tomentosa eggs in quarantine is also proposed.

斑马鱼系统中水气单胞菌、分枝杆菌和Pseudocapillaria 毛白杨的环境筛选

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

胞外酸性 pH 培养体系的建立

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...