Name: dissection and culture of chick statoacoustic ganglion and spinal cord explants in collagen gels for neurite outgrowth assays
Uploaded: 2011-12-20T13:00:00
Description: Watch this Scientific Journal Video about Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays at JoVE.com

这项工作是由国家卫生部批准RO1DC002756和普渡大学研究基金会。我们感谢与实验的意见和罗德尼帮助数字McPhail多丽丝吴和维泽张。

食谱小鸡林格<table border="1"><tbody><tr><td> 7.2摹</td><td>氯化钠</td></tr><tr><td> 0.23摹</td><td>氯化钙2 + 2H 2 O </td></tr><tr><td> 0.37摹</td><td>氯化钾</td></tr><tr><td> 0.115摹</td><td> NA 2 HPO 4 </td></tr><tr><td> 900毫升</td><td>水（组织文化品位） </td></tr></tbody></table>注：调整pH值至7.4。使终体积为1000毫升水。 10X PBS <table border="1">...

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我们提出了一个方法来剖析和文化的E4凹陷和E6脊髓植，从小鸡无血清条件下，。目前，这个过程是在我们的实验室研究SAG神经元的存活和突起生长分泌各种配体的影响。本议定书的小说方面，包括使用的无血清培养的外植体浸泡在生长因子的研究凹陷突起生长的影响的珠。珠的方法类似我们已经描述鸡胚脊髓 10 。传统上，珠已被用于研究与经典的轴突导向因子和morphogens。在这里，我们?...

<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> 设备 </td><td></td><td></td><td></td></tr><tr><td>解剖镜下</td><td></td><td></td><td>我们用明基地照明的Leica M80体视显微镜</td></tr><tr><td>滑入温暖</td><td> CS＆E </td><td></td><td>胶原蛋白的聚合</td></tr><tr><td>鸡蛋孵化器</td><td></td><td></td><td></td></tr><tr><td> 37 ° C / 5％CO 2培养箱细胞培养孵化器</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 夹层材料 </td><td></td><td></td><td></td></tr><tr><td> Sylgard＆副本;涂层的培养皿</td><td></td><td></td><td></td></tr><tr><td> 24孔细胞培养板</td><td>康宁</td><td> 3526 </td><td></td></tr><tr><td> ＃5杜蒙钳</td><td>精细科学工具</td><td> 11252-30 </td><td></td></tr><tr><td> ＃55杜蒙钳</td><td>精细科学工具</td><td> 11295-51 </td><td></td></tr><tr><td>不锈钢解剖引脚</td><td>精细科学工具</td><td> 26002-10 </td><td>胚胎穿针，精细解剖</td></tr><tr><td>莫里亚穿孔勺</td><td>精细科学工具</td><td> 10370-17 </td><td>胚胎收获/转让</td></tr><tr><td> 200μL宽口吸管提示</td><td>点科学公司</td><td> 118 - 96R </td><td>外植体转移</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> E4和E6鸡蛋</td><td></td><td></td><td> ＆NBSP; </td></tr><tr><td>林格小鸡的解决方案</td><td></td><td></td><td>胚胎收获</td></tr><tr><td> HBSS </td><td>西格玛</td><td> H8264 </td><td>凹陷清扫</td></tr><tr><td> L15的介质</td><td>西格玛</td><td> L5520 </td><td>脊髓夹层介质</td></tr><tr><td>胎牛血清</td><td>亚特兰大生物</td><td> S11150 </td><td>脊髓夹层介质</td></tr><tr><td> Vannas剪刀</td><td>世界精密仪器</td><td> 501778 </td><td>脊髓夹层</td></tr><tr><td>&nbsp;</td><td></td><td></td><td></td></tr><tr><td> 胶原蛋白的准备 </td><td></td><td></td><td></td></tr><tr><td>鼠尾I型胶原</td><td> BD公司</td><td> 354249 </td><td>植文化</td></tr><tr><td> 10X PBS（无菌） </td><td></td><td></td><td>为神经tralize胶原蛋白</td></tr><tr><td> 1N氢氧化钠（无菌） </td><td></td><td></td><td>为了抵消胶原蛋白</td></tr><tr><td>蒸馏水（无菌） </td><td></td><td></td><td>为了抵消胶原蛋白</td></tr><tr><td> pH值指标文件（6.0-8.1） </td><td>瓦特曼</td><td> 2629-990 </td><td>要检查胶原蛋白的pH值</td></tr><tr><td> 15 ml锥形管</td><td>点科学公司</td><td> 818 - PG </td><td></td></tr><tr><td> 500μL宽口吸管提示</td><td>点科学公司</td><td> 119 - R100 </td><td>吸管胶原蛋白</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 植文化 </td><td></td><td></td><td></td></tr><tr><td> DMEM/F12培养基</td><td>西格玛</td><td> D8437 </td><td>植文化介质</td></tr><tr><td>及其1 </td><td>西格玛</td><td> I2521 </td><td>中等补充</tD&gt; </tr><tr><td>青霉素链霉素</td><td> Invitrogen公司</td><td> 15140-122 </td><td>中等补充</td></tr><tr><td>睫状神经营养因子（大鼠） </td><td>西格玛</td><td> C3835 </td><td>中等补充</td></tr><tr><td> NT - 3（人） </td><td>西格玛</td><td> N1905 </td><td>中等补充</td></tr><tr><td>纯净鼠标Wnt5a </td><td> R＆D系统</td><td> 645 - WN - 010 </td><td></td></tr><tr><td>阿菲 - 凝胶蓝胶珠</td><td> Bio - Rad公司</td><td> 153-7302 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 免疫组化 </td><td></td><td></td><td></td></tr><tr><td>多聚甲醛</td><td>西格玛</td><td> P6148 </td><td>固定</td></tr><tr><td> PBS </td><td></td><td></td><td>冲洗</td></tr><tr><td> TRITON X - 100 </td><td>西格玛</td><td> T9284 </td><td>封闭液</td></tr><tr><td>钠叠氮化物</td><td>西格玛</td><td> S8032 </td><td>封闭液</td></tr><tr><td>小牛血清</td><td> Invitrogen公司</td><td> 16170-078 </td><td>封闭液</td></tr><tr><td>鼠单克隆IgG2b抗β微管蛋白1 +第二抗体</td><td>西格玛</td><td> T8535 </td><td>标签胞体和突起</td></tr><tr><td>的Alexa Fluor 488山羊抗鼠IgG2的B抗体</td><td> Invitrogen公司</td><td> A21141 </td><td>检测β微管蛋白的抗体</td></tr><tr><td>聚四氟乙烯微型抹刀</td><td> VWR </td><td> 57949-033 </td><td>以及推出胶原凝胶</td></tr></tbody></table>

dissection and culture of chick statoacoustic ganglion and spinal cord explants in collagen gels for neurite outgrowth assays

鸡内耳感觉器官外围过程statoacoustic节（SAG）的神经元支配。感觉器官支配取决于轴突的指导线索和生存因素位于沿轨迹的轴突生长和/或在其感觉器官目标2的组合。例如，一个典型的轴突导向信号通路，semaphorin neuropilin，功能产生的干扰misrouting 3奥迪轴突。此外，多种生长因子在内耳的感官指标，包括神经营养因子3（NT - 3）和脑源性神经营养因子（BDNF），表示已在转基因动物的操纵，再次领先misrouting凹陷轴突 4 。同样是这些分子促进体外 5,6和突起生长的小鸡凹陷神经元的生存。 在这里，我们描述和证明在体外的方法，我们目前正在ü唱鸡凹陷突起的可溶性蛋白，其中包括知名morphogens如Wnts，以及促进SAG突起生长和神经元的存活重要的生长因子的响应测试。利用该模型系统，我们希望得出结论分泌的配体可以发挥SAG神经元的存活和突起生长的影响。 凹陷外植体解剖胚胎第4天（E4），并在三维胶原凝胶中培养24小时无血清条件下。首先，突起的响应测试与蛋白培养基中培养的外植体。然后，询问是否分泌配体的点源可以对突起生长的方向影响，外植体共同培养蛋白包被的磁珠和珠本地促进或抑制生长的能力检测。我们还包括一个解剖示范（修改后的协议7）E6脊髓植文化。我们经常使用脊髓植确认生物活性的蛋白质和蛋白质浸泡珠，来验证物种小鸡组织凹陷植相同的培养条件下，交叉反应。这些方便快速筛选分子施加营养（生存）或热带（定向）凹陷神经元的影响，特别是在执行体内研究，在体外实验。此外，这种方法允许单个分子的无血清条件下的测试，具有较高的神经元的存活8。

Watch this Scientific Journal Video about Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays at JoVE.com

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

我们演示了如何解剖和文化小鸡E4 statoacoustic节和E6脊髓植。外植体无血清条件下24小时的三维胶原凝胶培养。突起的响应测试与生长因子补充介质与蛋白包被的磁珠。

小鸡Statoacoustic神经节和脊髓植在胶原凝胶夹层和文化突起生长实验

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

Research

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Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

一个在培养皿中的功能电机组：脊髓植和肌肉细胞共培养

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

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Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Chicken Embryo Spinal Cord Slice Culture Protocol

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Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

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The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

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完全性脊髓损伤和脑解剖协议后续Wholemount<em&gt;原位</em&gt;杂交苗种海七鳃鳗

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

对基因差异表达的RNA序列中电穿孔鸡胚脊髓分析

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

一个程序，植入脊柱商会纵<em&gt;在体内</em&gt;鼠标脊髓成像

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

脊髓神经元从新生小鼠中分离和培养

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

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Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...