Name: analysis of trunk neural crest cell migration using a modified zigmond chamber assay
Uploaded: 2012-01-19T12:00:00
Description: Watch this Scientific Journal Video about Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay at JoVE.com

我们特别感谢利诺金，史蒂夫古兹曼和Ujit的Satyarthi的，这种方法在开发过程中的技术援助。迈伦·霍桑，理查德·Spengel，罗伯托·罗哈斯加工室使用，提供了必要的技术援助。值得一提的是，罗伯托·罗哈斯图4。我们也感谢斯科特·弗雷泽发展的趋化试验前的宝贵意见。这项工作得到部分支持，NIH-MBRS分数5S06GM048680-13 MEDB和加州州立大学北岭分校研究生论文支持计划奖CW。

1。第1天：过夜培养于盖玻片上的躯干神经管分离<ol><li> 56小时的孵育小鸡的鸡蛋在38℃中取出的鸡蛋孵化，轻度喷用70％的乙醇，然后让他们干。将鸡蛋打碎成紫外线消毒的玻璃盘。 </li><li>从每个胚胎中提取蛋黄，将其放置在小鸡林格氏。通过第一弯剪刀切割周围血岛，然后，用生硬的镊子，挑胚胎的胚外膜，并将其放置在无菌塑料培养皿中含有小鸡林格氏液。 </li><li>隔离每个胚胎的树干修剪...

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	<li>Le Douarin, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+avian+embryo+as+a+model+to+study+the+development+of+the+neural+crest:+a+long+and+still+ongoing+story.">The avian embryo as a model to study the development of the neural crest: a long and still ongoing story.</a> Mechanisms of Development. 121, 1089-1102 (2004).</li><li>Baker, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neural Crest and Cranial Ectodermal Placodes. , (2005).</li><li>Gammill, L. S., Roffers-Agarwal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Division+of+labor+during+trunk+neural+crest+development.">Division of labor during trunk neural crest development.</a> Dev. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ability+of+polymorphonuclear+leukocytes+to+orient+in+gradients+of+chemotactic+factors.">Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors.</a> The Journal of Cell Biology. 75, 606-616 (1977).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chicken+embryo.">A series of normal stages in the development of the chicken embryo.</a> J. Morph. 88, 49-52 (1951).</li><li>Boyden, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemotactic+effect+of+mixtures+of+antibody+and+antigen+on+polymorphonuclear+leucocytes.">The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes.</a> The Journal of Experimental Medicine. 115, 453-466 (1962).</li><li>Davis, E. M., Trinkaus, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significance+of+cell-to+cell+contacts+for+the+directional+movement+of+neural+crest+cells+within+a+hydrated+collagen+lattice.">Significance of cell-to cell contacts for the directional movement of neural crest cells within a hydrated collagen lattice.</a> Journal of Embryology and Experimental Morphology. 63, 29-51 (1981).</li><li>Zicha, D., Dunn, G. A., Brown, A. 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在主干NCC的趋化研究已经证明了具有挑战性的一系列原因。主干的NCC的构成一个异构干细胞的人口将分化，如果培养长期的，因此，躯干NCC的，必须从主外植的中继级NT。传统的方法来研究均匀分布在体外的细胞群的趋化反应是难以在主干NCC的测试，因为他们首先需要的细胞是孤立的均匀再接种的趋室（例如，Boyden小室12）。为外植几十个NTS可能会被要求创建一个均匀分布的主干NCC?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> DMEM </td><td>欧米茄科学</td><td> DM-22 </td><td></td></tr><tr><td>青霉素链霉素解决方案中</td><td>欧米茄科学</td><td> PS-20 </td><td> 100X股权集中度</td></tr><tr><td> L-谷氨酰胺</td><td>欧米茄科学</td><td> GS-60 </td><td> 100X股权集中度</td></tr><tr><td>胎牛血清</td><td>欧米茄科学</td><td> FB-11 </td><td>地段＃105247（或其他相媲美） </td></tr><tr><td>修改Zigmond室</td><td>自制</td><td> N / A </td><td>水库容积：160微升EA;其他规格，请参阅图。 4，补充制造协议</td></tr><tr><td>细胞培养皿</td><tD&gt;丹维尔</td><td> T6040 </td><td> 40×10毫米</td></tr><tr><td>纤维连接蛋白</td><td> BD </td><td> 354008 </td><td> 10倍的股票准备在1毫升H 2 O和9毫升DMEM稀释1毫克FN </td></tr><tr><td>盖玻片</td><td>费舍尔</td><td> 12-548-B </td><td>预清洗; 22 x 22毫米</td></tr><tr><td> L15介质</td><td> Thermo Scientific的</td><td> SH30525.02 </td><td></td></tr><tr><td>凡士林</td><td>舒适</td><td> 011110794642 </td><td> 100％ </td></tr><tr><td>离心管</td><td> Biologix </td><td> 10-9152 </td><td> 15毫升</td></tr><tr><td>中性蛋白酶</td><td>电池系统</td><td> 4Z0-850 </td><td> 10X股权集中度</td></tr><tr><td>注射器</td><td> BD </td><td> 309602 </td><td> 1毫升</td></tr><tr><td>针</td><td> BD </td><td> 305127 </td><td> 25 G x 1.5英寸</td></tr><tr><td>的Alexa Fluor 488-IgM阳性</td><td>我nvitrogen </td><td> A21042 </td><td>股票是2毫克/毫升; 7摩尔的染料/摩尔IgM抗体</td></tr><tr><td>解剖镊</td><td> FST </td><td>杂项。 </td><td>杜蒙特＃5或55;直嘴;不锈钢或钛</td></tr><tr><td>钨针</td><td> N / A </td><td> N / A </td><td>首页放置在针座</td></tr><tr><td>钝钳</td><td>蒂曼</td><td> 160-18 </td><td>用于转移胚胎林格从蛋黄中的</td></tr></tbody></table>补充协议：制作改进的Zigmond厅请参阅图4作为参考下面的协议： <ol><li>购买一张3/16“厚的抛光亚克力（4.45 mm实际厚度）。 </li><li>使用一台锯，切割室，以粗糙的尺寸33.25毫米x64.57毫米中的空白过大的。这允许3.175毫米的额外的用于加工的材料。 </li><li>在六室设置空白本身。有了一个铣床和6.35毫米（1/4“）的端铣刀位，精加工的腔室的侧面到其精确的尺寸：30.07毫米x61.39毫米。 </li><li>腔室定位空白的铣床上并定位沿x和y轴与一个边器的中心的坯件，然后零的中心位置。 </li><li>采集腔室高度（z轴）的顶表面接触的端铣刀位和零的高度。 </li><li>使用3.91毫米（0.154“）的端铣刀位，偏移位3.03毫米沿x轴（正方向）为第一储室开始加工成2.84毫米的深度，同时沿y轴移动（正方向）至7.62毫米（0.300“），然后遍历至7.62毫米（0.300”），在相反的方向（负）的长度为15.24毫米（0.600“）的一个完整的水库。偏移比特沿x轴方向（负方向）至3.03毫米（0.119“），并重复同样的过程，所述第二储。 </li><li>在它的边缘放置室钻一个孔，用1.09毫米（0.043英寸）钻头（共4个），每个水库的水库在实验过程中加载介质腔侧连接端。 </li><li>在温暖的肥皂水中浸泡室，以帮助消除任何化学污染物。 </li><li>浸泡和冲洗腔室，以及在双蒸水中，以消除任何肥皂。该商会是现在已经准备好使用上述。 </li></ol>

analysis of trunk neural crest cell migration using a modified zigmond chamber assay

神经嵴细胞（NCCS）是一个流动人口的细胞存在于脊椎动物的发展，移民从背神经管（NT）后发生上皮-间质转化1,2。在EMT，NCC的长距离迁移，沿刻板的途径，直到他们达到他们的目标。 NCC的区分成为一个巨大的数组类型的细胞，包括神经元，神经胶质细胞，黑素细胞和嗜铬细胞1-3。 NCC的接触和认识到自己的正确的目标位置的能力是基本适当的主干NCC-源性成分结构的形成。因此，阐明机制的指导主干NCC迁移的问题具有十分重要的意义。大量的分子已被证明来指导NCC迁移4。例如，躯干NCC的Semaphorin，和ephrin和缝配体的5-8负指导线索，如被击退。然而，并非直到最近，已确定9的任何趋化因子的主干NCC的。 常规的体外方法研究的贴壁细胞的趋化行为的工作最好与永生化的，均匀分布的细胞，但更具挑战性的适用于某些初级最初缺乏均匀的分布和迅速分化（如NCC的）的干细胞培养。均匀化分布的主干NCC的趋化研究的一种方法是从小学NT植文化的主干NCC的隔离，然后抬起replate他们几乎100％汇合。然而，这种电镀的方法需要大量的时间和精力，到取出足够的细胞，是残酷的，分布躯干的NCC的在一个不同的方式，发现在体内条件。 在这里，我们报道了在体外的方法，是可以评估的趋化和其他迁徙的主干NCC的回应没有requirin嘎同质细胞分布。此技术利用延时成像伯，泰然自若改性Zigmond室（一个标准Zigmond室其他地方描述10）内的中继线净捐助国。通过暴露在培养的周边的中继线净捐助国到趋化梯度是垂直于他们的预测的自然方向性，由所施加的趋化梯度诱导洄游极性的改变可以被检测。这种技术是廉价的，需要只有两个NT外植体每重复处理的培养，避免恶劣的细胞起重（如胰蛋白酶消化），叶中继线NCC的在一个更相似的分布，在体内条件下，外植和实验之间的时间量减少（这可能会降低分化的风险），并允许时间的推移众多的候鸟特性的评价。

Watch this Scientific Journal Video about Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay at JoVE.com

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

取出的细胞迁移（躯干神经嵴细胞）的方法来分析。这种方法是廉价的，温柔的，和能够区分趋化学增活现象和其他因素的影响，如在主躯干神经嵴细胞培养的细胞 - 细胞相互作用所产生的迁徙极性的。

躯干神经嵴细胞迁移使用一个修改过的Zigmond室法分析

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Research

JoVE Journal

Neuroscience

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

小鼠神经嵴干细胞的培养鸡胚提取物的生产

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

科研

神经科学

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

成年鼠神经干细胞的分离和扩大使用神经干含量

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

建立小鼠胚胎神经干细胞培养神经球含量

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

神经细胞集落形成细胞分析检测，以区分善意的神经祖细胞的神经干细胞

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

从人类毛囊神经嵴干细胞的分离和培养的

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

跨物种移植神经嵴迁移和分化的分析

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

神经母细胞含量：从区分神经干细胞后代使用流式细胞仪，神经祖细胞的生成和富集含量

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Co-analysis of Brain Structure and Function using fMRI and Diffusion-weighted Imaging

使用功能磁共振成像和弥散加权成像脑结构与功能分析

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...