Name: a quantitative cell migration assay for murine enteric neural progenitors
Uploaded: 2013-09-18T15:15:00
Description: Watch this Scientific Journal Video about A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors at JoVE.com

我们感谢丹尼斯FLIPO用于图像处理和分析意见，戴维W在银鱼的实验室生成的Gata4p [5KB]-GFP的小鼠品系。研究在实验室皮隆是由CIHR，NSERC，FRQS和FRQNT资助。

伦理声明在老鼠的实验进行了以下的在医学研究中使用动物的照顾和处理动物护理指引加拿大议会。涉及动物的操纵协议已获魁北克蒙特利尔大学的伦理委员会（科米特Institutionnel去保护DES ANIMAUX;编号0512-R3-650-0513）。 1。胶原蛋白凝胶的制备在无菌的方式工作，在组织培养罩。 <ol><li>准备完整的5倍DMEM培养基（Eagle的修改必需培养基）?...

<ol>
	<li>Bronner, M. E., 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=Development+and+evolution+of+the+neural+crest:+An+overview.">Development and evolution of the neural crest: An overview.</a> Dev. Biol. 366 (1), 2-9 (2012).</li><li>Bergeron, K. F., Silversides, D. W., Pilon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+genetics+of+Hirschsprung's+disease.">The developmental genetics of Hirschsprung's disease.</a> Clin. Genet. 83 (1), 15-22 (2013).</li><li>Obermayr, F., Hotta, R., Enomoto, H., Young, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+developmental+disorders+of+the+enteric+nervous+system.">Development and developmental disorders of the enteric nervous system.</a> Nat. Rev. Gastroenterol. Hepatol. 10 (1), 43-57 (2012).</li><li>Sasselli, V., Pachnis, V., Bursn, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enteric+nervous+system.">The enteric nervous system.</a> Dev. Biol. 366 (1), 64-73 (2012).</li><li>Sanchez, M. P., Silos-Santiago, I., 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=Renal+agenesis+and+the+absence+of+enteric+neurons+in+mice+lacking+GDNF.">Renal agenesis and the absence of enteric neurons in mice lacking GDNF.</a> Nature. 382 (6586), 70-73 (1996).</li><li>Baynash, A. G., Hosoda, K., 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=Interaction+of+endothelin-3+with+endothelin-B+receptor+is+essential+for+development+of+epidermal+melanocytes+and+enteric+neurons.">Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons.</a> Cell. 79 (7), 1277-1285 (1994).</li><li>Chalazonitis, A., Pham, T. D., 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=Neurotrophin-3+is+required+for+the+survival-differentiation+of+subsets+of+developing+enteric+neurons.">Neurotrophin-3 is required for the survival-differentiation of subsets of developing enteric neurons.</a> J. Neurosci. 21 (15), 5620-5636 (2001).</li><li>Goldstein, A. M., Brewer, K. C., Doyle, A. M., Nagy, N., Roberts, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+signaling+is+necessary+for+neural+crest+cell+migration+and+ganglion+formation+in+the+enteric+nervous+system.">BMP signaling is necessary for neural crest cell migration and ganglion formation in the enteric nervous system.</a> Mech. Dev. 122 (6), 821-833 (2005).</li><li>Jiang, Y., Liu, M. T., Gershon, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netrins+and+DCC+in+the+guidance+of+migrating+neural+crest-derived+cells+in+the+developing+bowel+and+pancreas.">Netrins and DCC in the guidance of migrating neural crest-derived cells in the developing bowel and pancreas.</a> Dev. Biol. 258 (2), 364-384 (2003).</li><li>Ramalho-Santos, M., Melton, D. A., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signals+regulate+multiple+aspects+of+gastrointestinal+development.">Hedgehog signals regulate multiple aspects of gastrointestinal development.</a> Development. 127 (12), 2763-2772 (2000).</li><li>Natarajan, D., Marcos-Gutierrez, C., Pachnis, V., de Graaf, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+signaling+by+receptor+tyrosine+kinase+RET+for+the+directed+migration+of+enteric+nervous+system+progenitor+cells+during+mammalian+embryogenesis.">Requirement of signaling by receptor tyrosine kinase RET for the directed migration of enteric nervous system progenitor cells during mammalian embryogenesis.</a> Development. 129 (22), 5151-5160 (2002).</li><li>Young, H. M., Hearn, C. 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=GDNF+Is+a+chemoattractant+for+enteric+neural+cells.">GDNF Is a chemoattractant for enteric neural cells.</a> Dev. Biol. 229 (2), 503-516 (2001).</li><li>Amiel, J., Sproat-Emison, E., 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=Hirschsprung+disease,+associated+syndromes+and+genetics:+a+review.">Hirschsprung disease, associated syndromes and genetics: a review.</a> J. Med. Genet. 45 (1), 1-14 (2008).</li><li>Pilon, N., Raiwet, D., Viger, R. S., Silversides, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+pre-+and+post-gastrulation+expression+of+Gata4+within+cells+of+the+inner+cell+mass+and+migratory+neural+crest+cells.">Novel pre- and post-gastrulation expression of Gata4 within cells of the inner cell mass and migratory neural crest cells.</a> Dev. Dyn. 237 (4), 1133-1143 (2008).</li><li>Nagy, N., Goldstein, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelin-3+regulates+neural+crest+cell+proliferation+and+differentiation+in+the+hindgut+enteric+nervous+system.">Endothelin-3 regulates neural crest cell proliferation and differentiation in the hindgut enteric nervous system.</a> Dev. Biol. 293 (1), 203-217 (2006).</li><li>Nagy, A., Gertsenstein, M., Vintersen, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the mouse embryo: a laboratory manual. , 209-250 (2003).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat. Methods. 9, 671-675 (2012).</li><li>Byth, K. F., Thomas, 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=AZD5438,+a+potent+oral+inhibitor+of+cyclin-dependent+kinases+1,+2,+and+9,+leads+to+pharmacodynamic+changes+and+potent+antitumor+effects+in+human+tumor+xenografts.">AZD5438, a potent oral inhibitor of cyclin-dependent kinases 1, 2, and 9, leads to pharmacodynamic changes and potent antitumor effects in human tumor xenografts.</a> Mol. Cancer Ther. 8 (7), 1856-1866 (2009).</li></ol>

我们展示了如何我们的体外外植体培养技术可以用来精确地量化肠溶NCC迁移潜力的GDNF的存在。这种精确的定量是通过使用200微米厚的vibratome肠组织切片而不是大块的近似大小，如先前所描述11,12,15大大便利。的确，这使得我们能与细胞中一种高度可重现设定合理数量的工作。值得注意的是，荧光肠溶NCC的小肠从该外植体条带被切断的尾最区域内的均匀分布还允许从一个单一的肠道?...

神经嵴细胞（NCC）是胚胎发育过程中形成了许多衍生工具的瞬态细胞类型特有的脊椎动物。该细胞群出现在神经板，相邻的非神经外胚层1的边界。在神经胚形成，为神经板的地方NCC沿形成神经管背侧边缘弯曲。 NCC再经过上皮 - 间质转化，分离和迁移远离神经管。 NCC殖各个胚胎结构，包括消化道那里形成整个肠神经系统（ENS），神经节的互连网络中嵌入肠壁。由于最近检讨2,3，许多基因都参与了这个复杂结构的发展。 大多数ENS的是从迷走神经管（ 即约准后脑/脊髓边界）一个小水池NCC原始的派生4。这些神经祖细胞到达前肠围绕胚胎一天（五）9.0小鼠，然后尾端迁移肠道间质内，直到约e15.0拓殖整个胚胎肠。结肠的神经祖细胞的未成年子集也由骶NCC，其侵入后肠道的相反方向到盲肠4提供。既迷走神经和骶NCC需要多次迁移，增殖，存活和分化，促进线索，以确保完全形成ENS的。在这方面，动物模型 - 尤其是转基因小鼠 - 是有助的在几个必要的细胞外配体的鉴定：GDNF（神经胶质细胞源性神经营养因子），内皮素-3，神经营养素-3，骨形态发生蛋白（骨形态发生蛋白），netrin诱导，以及声波和印度刺猬（Shh和Ihh信号）5-10。其中，GDNF通过酪氨酸激酶跨膜受体RET（转染过程中整理的）信号被确认为日Ë最关键的途径的吸引力和NCC的指导，并在胚胎肠道。 GDNF是由肠道间质细胞分泌，并形成了一个时空方式控制rosrrocaudal梯度是直接chemoattractive到肠道NCC，其表达RET 11,12。 除其他功能外，ENS通过与肠壁平滑肌相互作用调节消化道内运动。在肠道导致先天性巨结肠的末端区域神经节的缺席：受影响区段的强直性收缩导致堵塞，消化材料的上游积累和肠道和腹部的大规模扩张。先天性巨结肠症发生大约在5,000一只活产婴儿。肠道NCC的rostro-尾迁移模式被认为是主要的促进因素先天性巨结肠症的病因。从迁移NCC和b的最后部分的源结肠，最远owel被殖民统治，是最容易受到ENS中形成的缺陷。按照肠道NCC迁移的关键作用，GDNF / Ret信号的干扰是先天性巨结肠13的主要已知的遗传原因。 为了更好地研究华北克拉通和ENS的发展，我们产生了转基因小鼠线-命名Gata4p [5KB]-GFP 14 -在这种迁徙NCC都贴有绿色荧光蛋白（GFP）。我们接下来完善的体外细胞迁移测定中，由其它组11,12,15适于从发表的作品，即现在允许在各种生长因子，如GDNF的存在肠溶NCC迁移潜在精确定量。

<table border="1">
	<tbody>
		<tr>
			<td>DMEM powder</td>
			<td>Wisent</td>
			<td>219-010-XK</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Bioshop</td>
			<td>SOB999</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr>
			<td>Steriflip vacuum filtration system (0.22 micron)</td>
			<td>EMD Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin solution, 100x</td>
			<td>Wisent</td>
			<td>450-201-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Wisent</td>
			<td>095-150</td>
			<td>High quality grade</td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>BD biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Bioshop</td>
			<td>SHY700</td>
			<td>Diluted from 10 N stock then sterile-filtered</td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Cedarlane</td>
			<td>CLCYT305</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 24-well Plate</td>
			<td>BD biosciences</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>Fisher Scientific</td>
			<td>089515</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri dish</td>
			<td>VWR</td>
			<td>89000-306</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>P5493</td>
			<td>Cell culture grade</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica</td>
			<td>M125</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Bioshop</td>
			<td>AGA001</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Feather</td>
			<td>21</td>
			<td></td>
		</tr>
		<tr>
			<td>All Purpose Instant Krazy Glue Pen</td>
			<td>Krazy Glue</td>
			<td>KG824</td>
			<td></td>
		</tr>
		<tr>
			<td>HM 650V Vibrating-Blade Microtome</td>
			<td>Thermo Scientific</td>
			<td>920110</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9564</td>
			<td></td>
		</tr>
	</tbody>
</table>

a quantitative cell migration assay for murine enteric neural progenitors

神经嵴细胞（NCC）是一个短暂的和多能细胞群源于背神经管和广泛地在整个发展脊椎动物胚胎迁移。除了提供周边神经胶质细胞和神经元，NCC生成的黑素细胞，以及大部分的颅面骨骼。 NCC迁移和分化是由它们的轴向原点沿神经管和他们接触到不同的区域外线索的组合控制。细胞外配体的这种贡献是特别明显的肠神经系统（ENS），神经节的一个复杂的相互连接的网络，本地控制（其中包括）肠道肌肉运动和肠蠕动的形成过程中。在吻端到尾鳍时尚 - - 准肠道的整个长度大多数ENS是从华北克拉通，为了殖民承接了漫长的旅程一个小的初始池派生。其中已知的多条信号通路影响肠道定植NCC，GDNF / Ret信号被认为是最重要的。事实上，RET配体GDNF的时空方式控制分泌肠道间质，主要是负责吸引和RET表达肠NCC指导，并在胚胎肠道。在这里，我们描述的体外细胞迁移测定，利用转基因小鼠系具有荧光标记的NCC，这使得在各种生长因子，包括GDNF的存在肠溶NCC迁移潜在精确定量。

下面的结果是代表什么可以与这里描述的（ 图1）的技术来获得的。使用生长因子（ 即 GDNF）刺激的表达GFP的肠溶NCC出肠道外植体并进入胶原凝胶（ 图2）的迁移。虽然一些小区出来的外植体在无生长因子，这些大多不是GFP标记和表示被动条目。有必要从胶原凝胶取出肠切片，以便记录的结果，因为这个组织仍然是人口稠密通过荧光细胞和本来隐藏的细胞趴在胶原?...

Watch this Scientific Journal Video about A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors at JoVE.com

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

我们提出了一个先体外后体内的细胞迁移测定法，允许在各种生长因子的存在下肠道神经嵴细胞迁移的潜在精确定量。

定量细胞迁移检测方法的鼠肠神经前体细胞

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

Research

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Neuroscience

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

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Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

科研

神经科学

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). ...

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

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 ...

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 ...

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 ...

An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

An Optimized Procedure for Fluorescence-activated Cell Sorting FACS Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

荧光激活细胞分选（FACS）自主神经祖细胞的分离，从胎鼠的内脏器官的优化程序

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

一个的趋电含量为神经前体细胞迁移的动力学分析外部施加直流电场

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...