Name: reactivation of demembranated cell models in chlamydomonas reinhardtii
Uploaded: 2022-05-06T15:00:00
Description: Watch this Scientific Journal Video about Reactivation of Demembranated Cell Models in Chlamydomonas reinhardtii at JoVE.com

本研究得到了日本科学促进会KAKENHI（https://www.jsps.go.jp/english/index.html）对N.U.（19K23758，21K06295）和K.W.（19H03242，20K21420，21H00420），Ohsumi Frontier Science Foundation（https://www.ofsf.or.jp/en/）到K.W.的资助，以及来自人类，环境和材料（http://alliance.tagen.tohoku.ac.jp/english/）与N.U.和K.W.的开放式创新动态联盟的支持。我们感谢筱原美雪女士（法政大学）在准备这些数字时给予的帮助。

本研究使用野生型 的莱因哈蒂衣原体菌株CC-125。CC-125从衣原体资源中心获得（见 材料表），并在20-25°C下维持在三乙酸酯磷酸盐（TAP）16，1.5%琼脂糖培养基上。
1. 细胞培养
<ol><li>在20-25°C下，在12小时/ 12小时明暗期（光照期的光照条件：~50μmol光子m−2 s−1白光）在TAP培养基16中培养莱因哈蒂衣?...

<ol>
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B., Plummer, J., Sander, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlamydomonas+flagellar+mutants+lacking+radial+spokes+and+central+tubules.+Structure,+composition,+and+function+of+specific+axonemal+components.">Chlamydomonas flagellar mutants lacking radial spokes and central tubules. Structure, composition, and function of specific axonemal components.</a> Journal of Cell Biology. 76 (3), 729-747 (1978).</li><li>R&#252;ffer, U., Nultsch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flagellar+coordination+in+Chlamydomonas+cells+held+on+micropipettes.">Flagellar coordination in Chlamydomonas cells held on micropipettes.</a> Cell Motility and the Cytoskeleton. 41 (4), 297-307 (1998).</li><li>Sale, W. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submicromolar+levels+of+calcium+control+the+balance+of+beating+between+the+two+flagella+in+demembranated+models+of+Chlamydomonas.">Submicromolar levels of calcium control the balance of beating between the two flagella in demembranated models of Chlamydomonas.</a> Journal of Cell Biology. 98 (1), 97-107 (1984).</li><li>Okita, N., Isogai, N., Hirono, M., Kamiya, R., Yoshimura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototactic+activity+in+Chlamydomonas+'non-phototactic'+mutants+deficient+in+Ca2+-dependent+control+of+flagellar+dominance+or+in+inner-arm+dynein.">Phototactic activity in Chlamydomonas 'non-phototactic' mutants deficient in Ca2+-dependent control of flagellar dominance or in inner-arm dynein.</a> Journal of Cell Science. 118, 529-537 (2005).</li><li>Hyams, J. S., Borisy, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+flagellar+apparatus+of+Chlamydomonas:+characterization+of+forward+swimming+and+alteration+of+waveform+and+reversal+of+motion+by+calcium+ions+in+vitro.">Isolated flagellar apparatus of Chlamydomonas: characterization of forward swimming and alteration of waveform and reversal of motion by calcium ions in vitro.</a> Journal of Cell Science. 33, 235-253 (1978).</li><li>Fujiu, K., Nakayama, Y., Yanagisawa, A., Sokabe, M., Yoshimura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlamydomonas+CAV2+encodes+a+voltage-+dependent+calcium+channel+required+for+the+flagellar+waveform+conversion.">Chlamydomonas CAV2 encodes a voltage- dependent calcium channel required for the flagellar waveform conversion.</a> Current Biology. 19 (2), 133-139 (2009).</li><li>Bessen, M., Fay, R. B., Witman, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+control+of+waveform+in+isolated+flagellar+axonemes+of+Chlamydomonas.">Calcium control of waveform in isolated flagellar axonemes of Chlamydomonas.</a> Journal of Cell Biology. 86 (2), 446-455 (1980).</li><li>Wakabayashi, K., King, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Chlamydomonas+reinhardtii+flagellar+motility+by+redox+poise.">Modulation of Chlamydomonas reinhardtii flagellar motility by redox poise.</a> Journal of Cell Biology. 173 (5), 743-754 (2006).</li><li>Saegusa, Y., Yoshimura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cAMP+controls+the+balance+of+the+propulsive+forces+generated+by+the+two+flagella+of+Chlamydomonas.">cAMP controls the balance of the propulsive forces generated by the two flagella of Chlamydomonas.</a> Cytoskeleton. 72 (8), 412-421 (2015).</li><li>Gorman, D. S., Levine, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+f+and+plastocyanin:+their+sequence+in+the+photosynthetic+electron+transport+chain+of+Chlamydomonas+reinhardi.">Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi.</a> Proceedings of the National Academy of Sciences of the United States of America. 54 (6), 1665-1669 (1965).</li><li>Takano, W., Hisabori, T., Wakabayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+estimation+of+cytosolic+ATP+concentration+from+the+ciliary+beating+frequency+in+the+green+alga+Chlamydomonas+reinhardtii.">Rapid estimation of cytosolic ATP concentration from the ciliary beating frequency in the green alga Chlamydomonas reinhardtii.</a> Journal of Biological Chemistry. 296, 100156 (2021).</li><li>Wakabayashi, K., Yagi, T., Kamiya, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2+-dependent+waveform+conversion+in+the+flagellar+axoneme+of+Chlamydomonas+mutants+lacking+the+central-pair/radial+spoke+system.">Ca2+-dependent waveform conversion in the flagellar axoneme of Chlamydomonas mutants lacking the central-pair/radial spoke system.</a> Cell Motility and the Cytoskeleton. 38 (1), 22-28 (1997).</li><li>Yueh, Y. G., Crain, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deflagellation+of+Chlamydomonas+reinhardtii+follows+a+rapid+transitory+accumulation+of+inositol+1,4,5-trisphosphate+and+requires+Ca2++entry.">Deflagellation of Chlamydomonas reinhardtii follows a rapid transitory accumulation of inositol 1,4,5-trisphosphate and requires Ca2+ entry.</a> Journal of Cell Biology. 123 (4), 869-875 (1993).</li><li>Wakabayashi, K., Ide, T., Kamiya, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-dependent+flagellar+motility+activation+in+Chlamydomonas+reinhardtii+in+response+to+mechanical+agitation.">Calcium-dependent flagellar motility activation in Chlamydomonas reinhardtii in response to mechanical agitation.</a> Cell Motility and the Cytoskeleton. 66 (9), 736-742 (2009).</li><li>Ueki, N., Wakabayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detergent-extracted+Volvox+model+exhibits+an+anterior-posterior+gradient+in+flagellar+Ca2.">Detergent-extracted Volvox model exhibits an anterior-posterior gradient in flagellar Ca2.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (5), 1061-1068 (2018).</li><li>Tanno, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+four-celled+Volvocales+green+alga+Tetrabaena+socialis+exhibits+weak+photobehavior+and+high-photoprotection+ability.">The four-celled Volvocales green alga Tetrabaena socialis exhibits weak photobehavior and high-photoprotection ability.</a> PLoS One. 16 (10), 0259138 (2021).</li></ol>

该协议中有两个关键步骤。第一种是称为去膜的过程，需要温和但彻底地进行。通过剧烈移液或涡旋诱导分化（即纤毛从细胞体中分离），即使在添加ATP后，细胞模型也无法移动。通常，将5×10个7 个细胞悬浮在〜0.5mL的去膜缓冲液中（最终细胞密度：1×108 个细胞/ mL）。如果细胞模型密度远低于此值（例如，1个×10个6 个细胞/mL），则细胞模型的再激活率将降低。这些细胞?...

去膜运动细胞的体外再活化是研究细胞运动调节机制的分子基础的宝贵工具。Szent-Györgyi首先通过添加三磷酸腺苷（ATP）1，首次证明了用50%甘油提取的兔骨骼肌纤维的体外收缩。这个实验是第一个证明ATP为肌肉收缩提供能量的实验。该方法很快被应用于ATP激励的睫状/鞭毛运动的研究，例如精子鞭毛2，纤毛副膜3和克莱因哈蒂环状衣原体（也称为鞭毛）4，使用非离子洗涤剂进行去膜化。
单细胞绿藻 C. reinhardtii 是研究纤毛的模式生物：它通过像人类的蛙泳一样击败它们来与两个纤毛一起游泳5。纤毛运动由动力蛋白驱动，动力蛋白是一种基于负端定向微管的运动蛋白6，7。睫状动力素可分为外臂动力蛋白和内臂动力蛋白。缺乏每种动力蛋白的突变体已被分离为具有不同运动异常的慢游突变体。对这些突变体的详细 体外 运动性分析显著推动了动力蛋白研究8.
自去膜化C. reinhardtii细胞（细胞模型）的体外再活化实验以来，利用该方法及其衍生物已经取得了许多重要发现。例如，在一系列Ca2 +缓冲液中重新激活细胞模型，表明9表明两个纤毛受亚微摩尔Ca2 +的不同调控，并且这种不对称的纤毛控制使C. reinhardtii10的光定向成为可能。此外，纤毛都显示从前向游动模式（称为非对称波形）到向后游动模式（称为对称波形，在细胞被光或机械电击时短时间内出现）的波形转换11，12。这种波形转换由亚毫摩尔Ca2+调节，其通过所谓的细胞核细胞核装置（包含两个纤毛，基底体，连接基体的结构以及细胞核的残余物）11或分离纤毛13的去膜化轴突的重新激活来显示。除Ca2 +外，氧化还原（还原氧化）是一种调节纤毛跳动频率的信号，这通过在含有不同比例的还原谷胱甘肽与氧化谷胱甘肽14的氧化还原缓冲液中重新激活细胞模型来显示。此外，环磷酸腺苷（cAMP）不对称地调节两个纤毛，这表现为用光裂笼笼cAMP15重新激活轴突。这些体外发现，结合遗传学发现，使人们更深入地了解了C. reinhardtii纤毛调节的分子机制。
此处描述了用于重新激活单元模型的协议。该方法很简单，允许各种修饰，并且可以应用于与纤毛一起移动的多个生物体。然而，由于去膜化细胞是脆弱的，因此需要一些技巧才能以良好的效率重新激活细胞模型的运动性，同时防止分升。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 mL plastic tube</td>
			<td>QSP</td>
			<td>502-PLN-Q</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>SARSTEDT</td>
			<td>62.554.502</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#39;-triphosphate disodium salt hydrate (ATP)</td>
			<td>Sima-Aldrich</td>
			<td>A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>KUBOTA</td>
			<td>2800</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlamydomonas strain CC-125</td>
			<td>Chlamydomonas Resource Center</td>
			<td></td>
			<td>https://www.chlamycollection.org/</td>
		</tr>
		<tr>
			<td>Creatine kinase</td>
			<td>Merck</td>
			<td>CK-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>Creatine phosphate</td>
			<td>Merck</td>
			<td>CRPHO-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Nakalai tesque</td>
			<td>14128-46</td>
			<td></td>
		</tr>
		<tr>
			<td>GEDTA(EGTA)</td>
			<td>Dojindo</td>
			<td>G002</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Dojindo</td>
			<td>GB70</td>
			<td></td>
		</tr>
		<tr>
			<td>Igepal CA-630</td>
			<td>Sigma-Aldrich</td>
			<td>I8896</td>
			<td>IUPAC name is octylphenoxypolyethoxyethanol: IGEPAL CA-630 is a substitute for Nonidet P-40 (NP-40); NP-40 is no longer available in Sigma-Aldrich.</td>
		</tr>
		<tr>
			<td>MgSO4-7H2O</td>
			<td>Nakalai tesque</td>
			<td>21002-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX-53</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>fisher scientific</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol, Mr 20,000</td>
			<td>Merck</td>
			<td>8.18897.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Pottasium acetate</td>
			<td>Nakalai tesque</td>
			<td>28434-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Nacalai</td>
			<td>31511-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>196-00015</td>
			<td></td>
		</tr>
	</tbody>
</table>

reactivation of demembranated cell models in chlamydomonas reinhardtii

自从Szent-Györgyi在20世纪中叶证明的通过添加ATP来收缩甘油化肌肉的历史实验以来，去膜化细胞的体外再激活一直是检查细胞运动的传统而有效的方法。这种实验方法的根本优点是再活化溶液的组成可以很容易地改变。例如，由于体内膜激发而仅暂时发生的高Ca2 +浓度环境可以在实验室中复制。真核纤毛（又名鞭毛）是精心设计的运动机制，其调节机制仍有待澄清。单细胞绿藻衣原体是纤毛研究领域的优秀模式生物。使用C. reinhardtii的去膜化细胞模型及其衍生物（例如分离的纤毛的去膜化轴突）的再激活实验对理解纤毛运动的分子机制有显着贡献。这些实验澄清了ATP为纤毛运动提供能量，并且各种细胞信号，包括Ca2 +，cAMP和活性氧，调节纤毛运动。这里描述了C. reinhardtii细胞的去膜和细胞模型的再激活的精确方法。

这里显示了 C. reinhardtii 野生型菌株（CC-125）的脱膜和再活化过程。接种后2天的培养物变成浅绿色（步骤1.1）（图1）。收集细胞（步骤2.1），洗涤（步骤2.2）和去膜（步骤2.5）。去膜后，所有细胞模型都变得不对称（步骤2.7）。去膜化的纤毛（称为轴突）仍然附着在细胞体上，这表明细胞模型的不动性不是由分化引起的（图4）。与再活化缓冲液...

Watch this Scientific Journal Video about Reactivation of Demembranated Cell Models in Chlamydomonas reinhardtii at JoVE.com

Reactivation of Demembranated Cell Models in Chlamydomonas reinhardtii

运动细胞的 体外 再激活是了解细胞运动机制的关键实验。该协议描述了重新激活 衣原体莱因哈蒂衣原体的去膜化细胞模型，这是一种研究纤毛/鞭毛的模式生物。

莱因哈蒂衣原体中去膜化细胞模型的再激活

Since the historical experiment on the contraction of glycerinated muscle by adding ATP, which Szent-Gy&#246;rgyi demonstrated in the mid-20th century, in ...

这种方法可以帮助了解哪些因素会影响纤毛运动。该技术的主要优点是，这种现象只在体内暂时发生，这种纤毛中钙离子浓度的反转可以在体外连续观察到。该方法特别有助于运动纤毛的研究领域。初学者很难执行去膜化步骤。因此，强烈建议您轻柔而精确地执行这些步骤。首先在20摄氏度下在1， 000 RCF下离心约10毫升莱因哈蒂衣原体培养物3分钟。倾析上清液，并用巴斯德移液器吸出残余物。将沉淀物重悬于约五毫升的洗涤缓冲液中。在洗涤缓冲液中以1， 000 RCF在20摄氏度下离心细胞3分钟。用移液管小心地丢弃上清液。向细胞沉淀中加入约0.5毫升去膜缓冲液。轻轻摇动试管以将细胞大致悬浮在缓冲液中，然后将试管放在冰上。用移液管轻轻悬浮剩余的细胞沉淀，然后再次将管子放在冰上。取5至10微升细胞模型，用稀释缓冲液稀释十倍，并在显微镜下观察以确认所有细胞模型均去膜化且不游泳。在0.5毫升管中，通过敲击管将80微升的再活化溶液，10微升的ATP溶液和10微升的电池模型混合。将大约30微升的混合溶液放在载玻片上，并用垫片轻轻地在其上放置盖玻片，以避免对细胞模型进行机械冲击。在显微镜下观察重新激活的细胞模型。在C.reinhardtii培养物脱膜后，所有细胞模型变得不育，去膜化的纤毛仍然附着在细胞体上，证实了细胞模型的不动性不是由去纤毛引起的。在ATP中将细胞模型与再活化缓冲液混合后，超过50%的细胞模型变得可运动。即使没有ATP再生系统，再活化的细胞继续移动约90分钟，最终ATP浓度为1毫摩尔，然后逐渐停止。去膜步骤至关重要。所有的细胞都应该停止游泳，但它们不应该混合得如此强烈，以至于发生去纤毛。研究人员可以修改该协议以包括钙离子或环AMP等试剂，或者对缺乏特定蛋白质的突变体进行实验以确定其生物学作用。

Research

JoVE Journal

Biology

Reactivation of Demembranated Cell Models in Chlamydomonas reinhardtii

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

交配和四分体分离衣藻遗传分析

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and ...

科研

生物学

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease

异位和原位气管移植小鼠模型研究闭塞性气道疾病的发展

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

视频人类胚胎干细胞克隆生长的生物信息学分析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Principles of Rodent Surgery for the New Surgeon

鼠类外科的原则，为新的外科医生

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform ...

Manual Restraint and Common Compound Administration Routes in Mice and Rats

手动约束和常见的化合物在小鼠和大鼠的给药途径

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

Evaluation of Mammary Gland Development and Function in Mouse Models

乳腺的发育和功能在小鼠模型的评价

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

剖检诊断，并选择在大鼠和小鼠的组织和样品收集

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

类固醇受体的生化重建•Hsp90的蛋白质复合物和配体激活绑定

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

精子冷冻保存和恢复使用I ·冷冻包

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models

旋转壁式生物反应器的培养和应用推导出三维上皮细胞模型

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. ...