Name: isolation and culture of neural crest cells from embryonic murine neural tube
Uploaded: 2012-06-02T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube at JoVE.com

我们要感谢马克·沃兹尼亚克视频援助。我们也想承认在得克萨斯大学西南肖恩·莫里森的培养大鼠数控细胞的原始协议。这项工作是支持范德比尔特大学医学中心的学术计划支持，由国立卫生研究院（HD36720，HD036720-11S109）和的果酸11GRNT7690040的为PAL，从AHA（0615209B）和国立卫生研究院（NS065604）博士前奖学金不结盟运动，和ERP赠款由美国国立卫生研究院培训资助T32HD007502支持。

1。准备板<ol><li>在任何时候使用无菌技术。 </li><li>纤维连接蛋白（FN）Dulbecco的PBS（DPBS）在3.3毫升的稀释成终体积100μL人体血浆FN的股票准备。终浓度为30微克/毫升，这可以在4°C保存1周。 </li><li>覆盖的底部，并让每个无菌组织培养与FN的解决方案4孔板以及坐了15分钟。确保覆盖整个表面。在这段时间内（步骤2和3）媒体。 </li><li>删除FN的解决方案，并让板干燥。轻轻冲洗井与500μL的DMEM培养液，...

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小心应注意的胚胎发育阶段，以确保这种方法的成功。计数小鼠早期胚胎的体节是匹配的胚胎内的枯枝落叶，并确定正确地区神经管隔离阶段的关键。一个或两个胚胎体节之间的变化是在合理的范围内的发展时机，取决于进行试验的决议。 DPC 9和9.5之间的胚胎将有17和25体节之间。如果胚胎拥有超过25体节，是进一步推进数控发育时间，数控副产物，将在文化，那么强劲。分期根据胚胎体节数，便?...

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DMEM培养基（低糖） </td><td> GIBCO / Invitrogen公司</td><td> 11885 </td><td></td></tr><tr><td>的Neurobasal中等</td><td> Gibco公司</td><td> 21103 </td><td></td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A3912-10G </td><td></td></tr><tr><td> DPBS </td><td> Gibco公司</td><td> 14190-144 </td><td></td></tr><tr><td> IGF1的</td><td> BD公司</td><td> 354037 </td><td>储存在50微克/毫升，分装在-20°C </td></tr><tr><td>碱性成纤维细胞生长因子</td><td> BD公司</td><td> 354060 </td><td>在25微克/毫升分装保存在-20°C </td></tr><tr><td>纤维连接蛋白</td><td> Gibco公司</td><td> 33016-015 </td><td>存储在为1mg/mL人iquots在-20°C。 </td></tr><tr><td>维甲酸</td><td>西格玛</td><td> R2625 </td><td>乙醇在-20°C重组后，在35微克/毫升分装店</td></tr><tr><td> 2 - 巯基乙醇</td><td>西格玛</td><td> D - 5637 </td><td></td></tr><tr><td> 氮的补充</td><td> Gibco公司</td><td> 17502-048 </td><td></td></tr><tr><td> B27的补充</td><td> Gibco公司</td><td> 17504-044 </td><td></td></tr><tr><td> steriflip 0.22微米的过滤器</td><td> Millipore公司</td><td> SCGP00525 </td><td></td></tr><tr><td>青霉素链霉素</td><td> Invitrogen公司</td><td> 15140122 </td><td></td></tr><tr><td> 0.20微米的过滤器</td><td>康宁</td><td> 431219 </td><td></td></tr><tr><td>注射器（过滤） </td><td> BD公司</td><td> 301604 </td><td></td></TR&gt; <tr><td>四孔板</td><td>赛默飞世尔科技</td><td> 176740 </td><td></td></tr><tr><td>胶原酶/ Dispase酶</td><td>罗氏公司</td><td> 269​​ 638 </td><td>活动由一批不同。储存在-20°C 100毫克/毫升等分</td></tr><tr><td>胰岛素针 （29半规） </td><td>贝克顿·迪克森</td><td> 309306 </td><td></td></tr><tr><td>缺氧商会</td><td>比卢普斯-罗滕伯格</td><td></td><td></td></tr><tr><td>氧分析仪</td><td>比卢普斯-罗滕伯格</td><td></td><td></td></tr><tr><td>镊子＃5 </td><td>罚款科学工具</td><td></td><td>为消除子宫和蜕膜。 </td></tr><tr><td>胰蛋白酶-EDTA（0.25％） </td><td> Gibco公司</td><td> 25200 </td><td></td></tr></tbody></table>

isolation and culture of neural crest cells from embryonic murine neural tube

胚胎神经嵴（数控）是一个多能祖人口，背神经管方面的，经历了一个上皮间质转化（EMT）和迁移整个胚胎，从而引起不同的细胞类型1-3。数控也有独特的能力，影响靶器官4-6的分化和成熟。 在体外 explanted时，数控祖细胞进行自我更新，迁移和分化成各种组织类型，包括神经元，神经胶质细胞，平滑肌细胞，软骨和骨。 数控多能首次描述7-9禽流神经管的外植体。数控细胞体外分离，有利于数控加工动力学研究，包括细胞增殖，迁移和多能。在禽流感和大鼠系统的进一步工作表明，explanted数控细胞保留其数控潜力，移植胚胎10时-13。因为这些固有的保存explanted数控祖细胞特性，神经管外植体检测提供了一个有吸引力的选择研究在体外数控。 为了达到更好地了解了哺乳动物的数控，许多方法已受聘隔离数控人口。数控源性祖细胞培养胚胎和成年后洄游数控祖11,14-20研究动态，可以从迁移后的位置然而数控祖细胞的分离，因为他们从神经管移民提供最佳的保护， NC细胞的潜力和洄游特性13,21,22。有些协议采用荧光激活细胞分类（FACS）来隔离丰富的NC为11,13,14,17尤其祖人口。然而，与早期胚胎开始时，细胞数量足够用于分析是很难获得与流式细胞仪，复杂的隔离早期数控人群的从单个胚胎的NS。在这里，我们描述的方法，不依赖流式细胞仪和结果约96％的纯数控1 WNT1-Cre重组酶激活血统记者23人口。 这里介绍的方法是改编从大鼠数控11,13文化优化的协议。以前的方法相比，这种协议的优点是：1）细胞生长饲养层，2）没有需要获得一个相对纯净的的数控人口，3）premigratory数控细胞分离和4流式细胞仪）的结果很容易量化。此外，该协议可用于数控隔离任何突变小鼠模型，促进数控特点与不同的基因操作的研究。这种方法的局限性是数控的胚胎，这是众所周知的影响数控2,24-28的存活，迁移和分化的背景下删除。

Watch this Scientific Journal Video about Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube at JoVE.com

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

胚胎神经嵴的神经管分离，便于使用<em&gt;在体外</em&gt;学习迁移，自我更新和多能神经嵴的方法。

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 ...

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

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

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

大鼠胚胎神经细胞的分离和培养：一个快速议定书“

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

从胚胎前脑内皮细胞的分离和培养

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

一个鼠标，两种文化：隔离和成人神经的培养干细胞从单个小鼠的两个神经源性区

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...