Name: preparation and morphological analysis of chick cranial neural crest cell cultures
Uploaded: 2022-06-27T13:30:00
Description: Watch this Scientific Journal Video about Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures at JoVE.com

我们感谢Corinne A. Fairchild和Katie L. Vermillion，他们参与了我们版本的小鸡颅神经褶皱培养方案的开发。

可以使用任何品种的 瘿 品种，包括白里格霍恩、金性链或罗德岛红。本研究中使用的鸡蛋品种多样，来源多样，包括当地农场和孵化场。
1. 溶液和材料的制备
<ol><li>通过混合 123.3 mM NaCl、1.53 mM CaCl 2、4.96 mM KCl、0.809 mM Na 2 HPO 4 和 0.147 mM KH2PO4 来制备林格氏溶液（参见材料表）。将pH调节至7.4，并将灭?...

<ol>
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L., Lidberg, K. A., Gammill, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+protein+methylation+is+essential+for+neural+crest+migration.">Cytoplasmic protein methylation is essential for neural crest migration.</a> Journal of Cell Biology. 204 (1), 95-109 (2014).</li><li>Yang, X., Li, J., Zeng, W., Li, C., Mao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elongator+Protein+3+(Elp3)+stabilizes+Snail1+and+regulates+neural+crest+migration+in+Xenopus.">Elongator Protein 3 (Elp3) stabilizes Snail1 and regulates neural crest migration in Xenopus.</a> Scientific Reports. 6 (1), 1-9 (2016).</li><li>Gonzalez Malagon, S. G., 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=Glycogen+synthase+kinase+3+controls+migration+of+the+neural+crest+lineage+in+mouse+and+Xenopus.">Glycogen synthase kinase 3 controls migration of the neural crest lineage in mouse and Xenopus.</a> Nature Communications. 9 (1), 1-15 (2018).</li><li>Bhattacharya, D., Azambuja, A. P., Simoes-Costa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+reprogramming+promotes+neural+crest+migration+via+yap/tead+signaling.">Metabolic reprogramming promotes neural crest migration via yap/tead signaling.</a> Developmental Cell. 53 (2), 199-211 (2020).</li><li>Jacques-Fricke, B. T., 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=Profiling+NSD3-dependent+neural+crest+gene+expression+reveals+known+and+novel+candidate+regulatory+factors.">Profiling NSD3-dependent neural crest gene expression reveals known and novel candidate regulatory factors.</a> Developmental Biology. 475, 118-130 (2021).</li><li>Bronner-Fraser, M., Garc&#237;a-Castro, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+4+manipulations+of+neural+crest+cells+or+their+migratory+pathways.">Chapter 4 manipulations of neural crest cells or their migratory pathways.</a> Methods in Cell Biology. 87, 75-96 (2008).</li><li>Milet, C., Monsoro-Burq, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+xenopus+laevis+neural+crest+for+in+vitro+explant+culture+or+in+vivo+transplantation.">Dissection of xenopus laevis neural crest for in vitro explant culture or in vivo transplantation.</a> Journal of Visualized Experiments. (85), e51118 (2014).</li><li>Malagon, S. G. G., 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=Dissection,+culture+and+analysis+of+primary+cranial+neural+crest+cells+from+mouse+for+the+study+of+neural+crest+cell+delamination+and+migration.">Dissection, culture and analysis of primary cranial neural crest cells from mouse for the study of neural crest cell delamination and migration.</a> Journal of Visualized Experiments. (152), e60051 (2019).</li><li>Theveneau, E., Mayor, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+delamination+and+migration:+From+epithelium-to-mesenchyme+transition+to+collective+cell+migration.">Neural crest delamination and migration: From epithelium-to-mesenchyme transition to collective cell migration.</a> Developmental Biology. 366 (1), 34-54 (2012).</li><li>Conrad, G. W., Bee, J. A., Roche, S. M., Teillet, 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=Fabrication+of+microscalpels+by+electrolysis+of+tungsten+wire+in+a+meniscus.">Fabrication of microscalpels by electrolysis of tungsten wire in a meniscus.</a> Journal of Neuroscience Methods. 50 (1), 123-127 (1993).</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+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Journal of Morphology. 88 (1), 49-92 (1951).</li><li>Gammill, L. S., Jacques-Fricke, B., 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=Embryological+and+genetic+manipulation+of+chick+development.">Embryological and genetic manipulation of chick development.</a> Methods in Molecular Biology. 1920, 75-97 (2019).</li><li>Vandekerckhove, J., Deboben, A., Nassal, M., Wieland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phalloidin+binding+site+of+F-actin.">The phalloidin binding site of F-actin.</a> The EMBO Journal. 4 (11), 2815-2818 (1985).</li><li>Bronner-Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+early+stages+of+trunk+neural+crest+migration+in+avian+embryos+using+monoclonal+antibody+HNK-1.">Analysis of the early stages of trunk neural crest migration in avian embryos using monoclonal antibody HNK-1.</a> Developmental Biology. 115 (1), 44-55 (1986).</li><li>Schindelin, J., 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=Fiji:+An+open-source+platform+for+biological-image+analysis.">Fiji: An open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Soille, P., Vincent, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+watersheds+in+digital+pictures+via+flooding+simulations.">Determining watersheds in digital pictures via flooding simulations.</a> Visual Communications and Image Processing '90: Fifth in a Series. (1360), 240-250 (1990).</li><li>Haupt, A., Minc, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+cells+sense+their+own+shape+-+mechanisms+to+probe+cell+geometry+and+their+implications+in+cellular+organization+and+function.">How cells sense their own shape - mechanisms to probe cell geometry and their implications in cellular organization and function.</a> Journal of Cell Science. 131 (6), (2018).</li><li>Ezin, M., Fraser, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+11+time-lapse+imaging+of+the+early+avian+embryo.">Chapter 11 time-lapse imaging of the early avian embryo.</a> Methods in Cell Biology. 87, 211-236 (2008).</li><li>Kulesa, P. M., Bailey, C. M., Cooper, C., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+ovo+live+imaging+of+avian+embryos.">In ovo live imaging of avian embryos.</a> Cold Spring Harbor Protocols. 5 (6), (2010).</li><li>McKinney, M. C., Kulesa, 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=Live+imaging+of+the+neural+crest+cell+epithelial-to-mesenchymal+transition+in+the+chick+embryo.">Live imaging of the neural crest cell epithelial-to-mesenchymal transition in the chick embryo.</a> Methods in Molecular Biology. 2179, 107-114 (2021).</li><li>Gustafson, C. M., Roffers-Agarwal, J., Gammill, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+cranial+neural+crest+cells+release+extracellular+vesicles+that+are+critical+for+their+migration.">Chick cranial neural crest cells release extracellular vesicles that are critical for their migration.</a> Journal of Cell Science. , (2022).</li><li>Williams, R., Sauka-Spengler, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+ovo+electroporation+of+early+chicken+embryos.">Ex ovo electroporation of early chicken embryos.</a> STAR Protocols. 2 (2), 100424 (2021).</li><li>Moulton, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+morpholinos+to+control+gene+expression.">Using morpholinos to control gene expression.</a> Current Protocols in Nucleic Acid Chemistry. 68 (1), 4-30 (2017).</li><li>Gandhi, S., 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=A+single-plasmid+approach+for+genome+editing+coupled+with+long-term+lineage+analysis+in+chick+embryos.">A single-plasmid approach for genome editing coupled with long-term lineage analysis in chick embryos.</a> Development. 148 (7), (2021).</li><li>Williams, R. 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这里描述的技术提供了一种适应性强的方法来分离雏鸡神经褶皱并对其进行铺板以创建迁徙性颅 NCC 培养物。这些培养物为轻松分析雏鸡NCC迁移和形态提供了简化的2D条件，可以补充卵巢成像方法中更具技术挑战性的24，25，26。虽然这种体外方法相对简单，但一致的结果取决于高质量的卵子和试剂。此外，由于培?...

神经嵴细胞（NCC）是脊椎动物胚胎中的瞬时细胞群。NCC 指定在神经板的边界，并经历上皮到间充质的过渡 （EMT） 以从背神经管迁移1。EMT后，NCC广泛分散在整个胚胎中，最终分化并促成各种结构，包括颅面骨骼，心脏流出道和大部分周围神经系统2。细胞极性、细胞骨架和粘附特性的变化是这种从迁移性细胞群向迁移性细胞群转变的基础3.研究NCC EMT和迁移提供了对细胞运动基本机制的见解，并为预防和治疗出生缺陷和癌症转移的努力提供了信息。
虽然体内分析对于理解胚胎环境中的NCC发育过程至关重要，但体外方法提供了视觉和物理可及性，促进了其他实验途径。在简化的 2D 环境中，可以评估 NCC 形态、细胞骨架结构和迁移距离。此外，遗传或可溶性因子扰动对运动NCC迁移行为的影响可以分析4，5，6，7，8，9，10。此外，可以收集、汇集分离的迁徙或迁移性 NCC 并用于高通量方法，以通过蛋白质组、转录组和表观基因组分析研究 NCC 的发育调控7，11。虽然有方法可用于从各种发育模式生物制备颅骨NCC12，13，14，但本文展示了那些首次学习从鸡胚胎培养颅骨NCC的方法的机制。
目前的协议描述了一种用于制备鸡颅NCC培养物的通用技术（图1）。由于NCC很容易从外植的神经褶皱迁移到培养基质上，因此雏鸡NCC自然地与胚胎组织分离，并且很容易产生原代培养物。随着中脑NCC从颅神经褶皱中大量迁移（与躯干15中长时间的逐细胞分层相反），这些培养物主要由迁移性颅神经嵴细胞组成，初始神经褶皱切除为迁移性NCC提供了一种收集方法。详细介绍了解剖和培养鸡颅神经褶皱的基本方法，并提供了该方法的不同应用和变化的建议。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63799/63799fig01v2.jpg"> 图 1：小鸡颅神经褶皱培养方案的示意图。 （A，B）从具有五个体细胞的雏鸡胚胎中切除颅神经褶皱（以蓝色轮廓显示）（如A的背视图所示）。灰色带，心脏新月形。（C）当接种在纤连蛋白上时，迁移性神经嵴细胞从神经褶皱中出现并分散到基质上。 <a href="https://www.jove.com/files/ftp_upload/63799/63799fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AxioObserver equipped with an LSM710 confocal scan head controlled by ZEN 3.0 SR software&#160;</td>
			<td>Zeiss</td>
			<td></td>
			<td>Used alpha Plan-Apochromat 100x/1.46 Oil DIC M27 objective</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Chamber dishes (glass bottom, single or divided)</td>
			<td>MatTek; Cell Vis</td>
			<td>P35G-1.5-14-C (MatTek) X000NOJQGX (Cellvis) 
			X000NOK1OJ (Cellvis)</td>
			<td>Single chamber 35 mm or 4 chamber 35 mm</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Carolina Biological Supply Company</td>
			<td>633029, 633031, 633033, 633035, 633037</td>
			<td>circles, 0.13&#8211;0.17 mm thickness, available in 12-25 mm diameter&#160;</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>ThermoFisher Scientific</td>
			<td>11320033</td>
			<td>Alternative for L15 media</td>
		</tr>
		<tr>
			<td>Egg incubator</td>
			<td>Sportsman</td>
			<td>1502</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS&#160;</td>
			<td>Life Technologies</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Fisher Scientific</td>
			<td>CB-40008A</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Whatman</td>
			<td></td>
			<td>grade 3MM chromatography</td>
		</tr>
		<tr>
			<td>Forceps (blunt)</td>
			<td>Fisher Scientific; Thomas Scientific</td>
			<td>08-890 (Fisher);1141W97 (Thomas)</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps (fine)</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td>Dumont #5</td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>https://fiji.sc/</td>
			<td></td>
			<td>Free image analysis software</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr>
			<td>L15 media</td>
			<td>Invitrogen</td>
			<td>11415064</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Media (Vectashield or ProLong Gold)</td>
			<td>Vector Laboratories; Thermofisher Scientific</td>
			<td>H-1700 (Vectashield); P36930 (ProLong Gold)</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-148</td>
			<td>10,000 Units/mL Penicillin; 10,000 mg/mL Streptomycin</td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>VWR (or similar)</td>
			<td></td>
			<td>60 mm, 100 mm</td>
		</tr>
		<tr>
			<td>Phalloidin</td>
			<td>Sigma-Aldrich</td>
			<td>P1951</td>
			<td>multiple flurophores available</td>
		</tr>
		<tr>
			<td>Pin holder</td>
			<td>Fine Science Tools</td>
			<td>26016-12</td>
			<td>For tungsten needle (alternative for spring scissors)</td>
		</tr>
		<tr>
			<td>Scissors (dissection)</td>
			<td>Fine Science Tools</td>
			<td>14061-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td>2.5 mm cutting edge (alternative for tungsten needle)</td>
		</tr>
		<tr>
			<td>Sylgard</td>
			<td>Krayden</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filters</td>
			<td>Sigma-Aldrich</td>
			<td>SLGVM33RS</td>
			<td>Millex-GV Syringe Filter Unit, 0.22 &#181;m, PVDF, 33 mm, gamma sterilized</td>
		</tr>
		<tr>
			<td>Tissue culture dishes</td>
			<td>Sarstedt</td>
			<td>83-3900</td>
			<td>35 mm culture dishes for bulk neural fold cultures</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten wire</td>
			<td>Variety of sources</td>
			<td></td>
			<td>0.01&#34; diameter for tungsten needle (alternative for spring scissors)</td>
		</tr>
	</tbody>
</table>

preparation and morphological analysis of chick cranial neural crest cell cultures

在脊椎动物发育过程中，神经嵴细胞（NCC）广泛迁移并分化成各种细胞类型，这些细胞类型有助于颅面骨骼和周围神经系统等结构。虽然在3D胚胎的背景下了解NCC迁移至关重要，但在2D培养中分离迁移细胞有助于可视化和功能表征，补充胚胎研究。本协议展示了一种分离鸡颅神经褶皱以产生原代NCC培养物的方法。迁移性NCC来自接种在纤连蛋白包被底物上的神经折叠外植体。这导致分散的、粘附的NCC群体，可以通过染色和定量形态学分析进行评估。这种简化的培养方法适应性强，可以与其他技术结合使用。例如，NCC迁移和迁移行为可以通过延时成像进行评估，或者通过包括抑制剂或基因表达的实验操作（例如，DNA，吗啉诺或CRISPR电穿孔）来询问功能。由于其多功能性，该方法为研究颅骨NCC的发展提供了一个强大的系统。

本协议的概述如图 1所示。打开孵化的鸡蛋，通过轻轻倒入戴手套的手掌中来分离带有胚胎表面的蛋黄（图2A，B）。清除白蛋白后（图2C），将滤纸框架涂在胚胎周围的蛋黄膜上，以促进切割和提起胚胎从蛋黄中取出，一旦卵黄膜被切割，蛋黄就开始溢出（图2D，E）。
<p class="jove_...

Watch this Scientific Journal Video about Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures at JoVE.com

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

这种多功能方案描述了通过从鸡胚胎中切除颅神经褶皱来分离前迁移神经嵴细胞（NCC）。在铺板和孵育时，迁移性NCC从神经折叠外植体中出现，允许在简化的2D环境中评估细胞形态和迁移。

鸡颅神经嵴细胞培养物的制备及形态学分析

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like ...

神经嵴细胞从胚胎中的颅神经褶皱迁移或放置在培养物中时。这提供了一种无需细胞解离即可分离原代迁移神经嵴细胞的便捷方法。虽然神经嵴迁移通常发生在三维胚胎环境中，但二维培养能够可视化和研究与迁移相关的过程。尽管存在颅神经折叠培养方案，但这种方法需要训练和生物体特异性步骤。该视频演示了促进鸡颅神经褶皱培养物可重复制备的技术。初学者经常难以解剖和电镀神经褶皱并评估培养结果。遵循解剖指南并应用此处描述的形态测量分析可以获得一致的定量结果。首先，将受精卵直立放置在 38 摄氏度的加湿培养箱内。从孵化器中取出鸡蛋后，彻底喷洒 70% 乙醇并使其干燥，对蛋壳进行消毒。为了固定和染色培养物，使用放入多孔组织培养皿中的玻璃盖玻片，并移取足够的纤连蛋白溶液以覆盖孔底。接下来，盖上盖子，将板与纤连蛋白溶液在 38 摄氏度的加湿培养箱中孵育至少一小时，同时解剖神经褶皱。用钝镊子在蛋壳上戳一个小孔，长度为鸡蛋长度的 1/4 到 1/3。小心地将钝镊子插入小孔中，确保不会破坏蛋黄。然后，切开鸡蛋周围的蛋壳，去除蛋壳的顶部，并将胚胎定位以进行隔离。使用闭合的钝钳的平坦边缘轻轻擦去覆盖胚胎的蛋黄表面上的任何多余白蛋白，以准备胚胎。使用镊子将滤纸支撑架放在胚胎上，胚胎在框架的窗口中。轻轻按下滤纸以粘附在蛋黄上。用解剖剪刀在滤纸框架的外侧剪开。用镊子或剪刀尖抓住框架的边缘，轻轻地将胚胎从蛋黄上抬起。将胚胎与纸框面朝下放入装有带笔链球菌的林格氏培养皿中。如果收集胚胎用于RNA或蛋白质敏感的下游应用，请将胚胎放在冰上。将胚胎转移到含有林格笔链球菌溶液的干净培养皿中。轻轻地来回漱口胚胎以清除任何遮挡视线的蛋黄，并更换林格氏笔链球菌溶液，或者在它变得浑浊时将其转移到新鲜的盘子中。将胚胎背侧和框架面朝上放置在解剖显微镜下。将胚胎留在纸架上，使其拉伸并保持到位。然后，去除卵黄碱膜，用镊子暴露神经褶皱。包括组织尾部至扩张的视囊泡和喙部至后脑，其中菱形收缩刚刚开始出现。使用弹簧剪刀，小心地切除中脑神经褶皱。注意切除神经褶皱的最背侧，尽量减少对神经管和非神经外胚层的污染，并使用 P20 移液器或无菌玻璃巴斯德移液管将神经褶皱转移到含有林格氏笔链球菌溶液的干净培养皿中，用卵黄林格笔链球菌溶液冲洗。将收集的褶皱存放在冰上，同时解剖其他褶皱。从培养箱中取出培养皿后，使用移液器或巴斯德移液器从盖玻片、培养皿或孔中取出纤连蛋白溶液。用林格笔链球菌溶液冲洗纤连蛋白包被的底物后，将适量的完整培养基添加到培养皿或孔中。为了阻挡塑料并防止纸巾粘连，请使用 P20 或 P200 移液器，并用蛋黄林格笔链球菌溶液冲洗移液器吸头。然后，将分离的神经褶皱转移到纤连蛋白包被的盖玻片的中心，注意尽可能少地转移林格氏笔链球菌溶液。让外植体沉淀 10 到 15 分钟后，通过缓慢而小心地携带带有镀神经褶皱的培养皿，将它们放入 38 摄氏度的加湿室中。用巴斯德移液管取出培养基，并用过滤灭菌的PBS冲洗孔。加入4%多聚甲醛，在室温下将其放在平台摇床上15分钟。孵育结束时，除去多聚甲醛，用PBS冲洗孔三次，并加入含有5%血清的PBST。现在，将 30 微升 200 纳摩尔俄勒冈绿共轭鬼笔环肽移液到每个盖玻片的光滑表面上。使用一对镊子，从含有5%血清的PBST上取下盖玻片，确保细胞朝上的方向。将盖玻片的边缘短暂接触到精致的任务刮水器上，以吸掉多余的液体。轻轻地将每个盖玻片面朝下放在稀释的鬼笔环肽滴上，并在室温下孵育30分钟。孵育期结束后，从染色溶液中提起盖玻片并将其放回培养皿中，将其翻转，使细胞侧朝上。确保用PBST盖住盖玻片，并将其放在平台振动器上10分钟，保持盖玻片盖住并在黑暗中。取出 PBST，然后重复清洗盖玻片两次，总共清洗三次 10 分钟。将一滴封片剂放在显微镜载玻片上。通过将盖玻片以一定角度缓慢降低到安装介质上，以避免产生气泡，并在成像前让盖玻片面朝下安装，并让介质在成像前凝固。最后，对染色的细胞进行成像，并将其导出为TIFF文件。神经嵴细胞在孵育后三到四个小时内开始从贴壁神经折叠外植体中出现，并在大约20小时后完成迁移。使用HNK-1标记迁移神经嵴细胞，大多数细胞似乎是HNK-1阳性。通过用鬼笔环肽染色丝状肌动蛋白来观察迁移的神经嵴细胞。使用ImageJ处理鬼笔环肽染色细胞的图像以产生阈值图像，其中细胞显示为黑色，背景为白色。分析阈值图像，并以对比条形图或小提琴图显示各种测量值。例如，分析的69个细胞的平均面积约为802.11平方微米，范围从60.27到2， 664.53平方微米。圆度反映了细胞的突起度，范围从 0.101 到 0.875。较低的值表示细长的形状，而值 1 表示完美的圆形。大多数细胞表现出细长的形状，这在小提琴图中最容易看到。该领域的迁移性神经嵴细胞的平均长宽比约为2.13，长宽比为1表示对称形状。仔细切除和外植体板对于成功的培养至关重要。让神经褶皱面朝下沉降 10 到 15 分钟，然后慢慢转移到培养箱中以鼓励粘附。这些技术的变化包括培养前的瞬时遗传操作和孵育期间的延时成像。此外，该协议允许收集迁移和迁移神经嵴细胞群以进行组学水平分析。

Research

JoVE Journal

Developmental Biology

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

一个人类神经干细胞系的三维文化，微小RNA的分析和推测的靶基因分化

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

科研

发育生物学

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

使用小鸡GFP 神经管移植和碳水化合物染料 DiI 注射，在鸡胚胎内对神经囊细胞和血管进行双重标记

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

交付<em&gt;在体内</em&gt;急性间歇性缺氧新生儿鼠害总理下区源性神经祖细胞培养

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to ...

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice ÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Â A High-throughput Screen in ES Cells

正向遗传学的方法来揭开抗逆基因小鼠 - 在ES细胞高通量筛选

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

转基因器官皮肤培养使用灭活人真皮代

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

培养人多能性和神经干细胞的基本和临床前研究一个封闭的细胞培养系统

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

从非转化人成纤维细胞增生四倍体细胞的建立

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

多光子时间推移成像实时可视化发展： 迁移神经嵴细胞在斑马鱼胚胎中的可视化

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

人胎脑神经干细胞 Zika 病毒感染的免疫细胞化学分析

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

发展中雏鸡光学顶盖中切向细胞迁移的可视化

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...