我们感谢桑德拉巴根出色的技术支持。这项工作是支持的蛋白质研究部（下称"珠三角"，总磷A1.2（RC和TS），擦研究情有独钟，Rektoratsprogramme - wissenschaftlicher Nachwuchs（AK）单克隆抗体39​​.4D5和F55A10发展研究杂交瘤细胞银行（DSHB，爱荷华城，IA）。

对于特殊的试剂:见标签。 1。列出的所有其他试剂在细胞培养级自Sigma Aldrich获得质量。 1。 PORN-H/laminin涂层菜/盖玻片<ol><li>准备工作0.5 mg / ml的聚- DL -鸟氨酸氢溴酸（色情- H）的解决方案，在150毫米硼酸盐缓冲液的pH值8.35。 <ol><li>一个50毫克色情/ 1毫升硼酸盐缓冲液的原液，可以提前做好准备，并储存在-20 ° C </li></ol></li><li>燃烧100％的乙醇消毒盖玻片，让它们风干。 </li><li>盖表​​面有足够的色情- H解决方案，在4一夜之间° C。 </li><li>次日用消毒的水和空气干燥的盖玻片三次。 </li><li>准备工作2.5微克/毫升层粘连蛋白的解决方案中的HBSS <ol><li>等分可以储存在-20 ° C解冻在4 ° C使用前。 </li></ol></li><li>与层粘连蛋白的解决方案色情H -涂层的盖玻片覆盖面，并让他们直到使用温度在室温孵育至少2小时。 </li></ol> 2。净化板的外源凝集素涂层<ol><li>准备无菌水，pH值9.5的10毫米三的解决方案。 </li><li>添加外源凝集素（解决的凝集素（Sigma公司L9640）的HBSS粉）至终浓度为10微克/毫升。 </li><li>涂层表面10厘米细胞培养皿中的凝集素的解决方案8毫升，让它在室温下至少30分钟孵育。 </li><li>洗板三次，直到使用的HBSS的HBSS和存储。 </li><li>准备一个30mm的氯化钾，0.8％（W / V）NaCl的去极化的解决方案。存储在室温（RT）的过滤和消毒。 </li></ol> 3。运动神经元文化的介质<ol><li>解冻马血清4℃过夜° C和灭活，在55℃，分装于-20℃到5毫升和存储30分钟° C。解冻等份直接使用前在室温下。 </li><li>储存在-20 ° C 1毫升aliqouts B27的补充使用前在室温下解冻B27的补充。避免冻融循环。 </li><li>分装到0.5毫升分装保存在-20 ° C glutamax，并用它在1:100。 </li><li>准备的10微克/毫升CNTF的0.1％BSA的水消毒的原液。储存在-20 ° C。 </li><li>混合B27的补充和直接使用前glutamax 0.5毫升，1毫升2.5毫升马血清46毫升neurobasal中等。 </li><li>睫状神经营养因子添加到终浓度为10毫微克/毫升培养基和预温介质为37 ° C </li></ol> 4。脊髓夹层<ol><li>牺牲一个E12.5孕鼠，并仔细取出胚胎。补充足够的RT的HBSS完全覆盖的胚胎。 </li><li>取出的头部和尾部的地方跨越四肢胚胎背面。 </li><li>修正了一个钳胚胎，并与其他镊子除去皮肤外。 </li><li>下刺入镊子取出脊髓和解除脊髓双方拉锯样动作。 </li><li>孤立的脊髓转移与新菜的HBSS和开放的脊髓背侧的中央通道。 </li><li>删除通过消除封闭脑膜脊髓背根神经节。 </li><li>收集高达6％凝集素板胚胎在充满1毫升的HBSS，并存储在冰上的Eppendorf反应管，直到编制完成的脊髓腰部分。 </li></ol> 5。基于凝集素的纯化富集的运动神经元注意:在开始的下一步骤之前，最新在这里准备和prewarm中期（见第3步。） <ol><li>准备一个胰蛋白酶的解决方案，解决1克在100毫升的HBSS，储存在-20 ° C和解冻胰蛋白酶在4 ° C </li><li>混合1G胰蛋白酶抑制剂，与98毫升的HBSS和2毫升1 M羟乙基pH值7.4，1毫升分装应保存在4 ° C </li><li>转移与细胞培养罩的脊髓管，小心取出700μL的HBSS。 </li><li>添加7.5μL胰蛋白酶溶液和混合仔细反相管。 </li><li>执行trypsination 8分钟，在37 ° C。 </li><li>停止加入30μl的胰蛋白酶抑制剂的反应和吸管达（磨碎）1000μL枪头小心10-15次，直到没有更多的细胞聚集是可见的。一个20-200μL移液器和相应的提示重复trituration。 </li><li>移液器到的HBSS充满凝集素板的电池解决方案，并轻轻转动板，分散细胞。 </li><li>请在室温下60分钟无振动的表面上的外源凝集素板。掩盖它，以避免污染。 </li><li> HBSS中轻轻取出，洗板与预先加热的HBSS仔细4次，以去除细胞碎片和不附加/独立的细胞。 </li><li>最后的洗涤步骤后，立即加入500μl的去极化解决方案板，让它孵化1分钟。 </li><li>促进支队的凝集素的约束细胞，并通过晃动敲击板。 </li><li> 2毫升预热的培养基，并转移到一个15毫升的猎鹰管填充板。 </li><li>与纽鲍尔计数室计数细胞数。 </li><li>板的细胞层粘连蛋白涂层的盖玻片上或在一个适当的数量取决于应用的板。 </li><li>执行第1天，随后每小心更换旧媒介的50％的第二天换液。始终使用预热，新鲜配制新的培养基。 </li></ol> 6。代表性的成果: 从小鼠胚胎胚胎运动神经元可在E12，E14。腰椎脊髓细胞总人口的2-3％由运动神经元和背根神经节神经元p75 的正常贸易关系的积极，重要的是要摆脱这些细胞开始之前基于凝集素preplating程序（的概述，请参阅图1和图2）。其次，脑膜包括强烈的分裂细胞，应排除为好，因为他们不能得到妥善的外源凝集素为基础的preplating程序（图2和图3）删除。更差的情况下，这些细胞可长满培养板。图3B和3D的有代表性的图片也给洗衣机已执行的程序后，对细胞密度较低的印象（见5.9协议的步骤）。通常保持了长达5年或7天的小鼠胚胎运动神经元的文化。在这段时间内的细胞建立其突起的最大长度（图4）。突起长度在很大程度上取决于基材板8。典型的基板层粘连蛋白，这是色情- H的有盖培养皿涂层。运动神经元也可以生长出其他或更少的基板上，像水裂解 8产生的细胞外基质。在不理想的涂层，突起长度和生长锥区情况下减少和细胞死亡通常会增加。营养的支持起到了文化的小鼠胚胎运动神经元生存至关重要的作用。最初七天营养支持6到10％至20％的下降没有镀细胞的生存。脑源性神经营养因子（BDNF）和睫状神经营养因子（CNTF）是最常用的因素，但其他人可以推动以及[白血病抑制因子（LIF），心肌营养素1（CT - 1），碱性成纤维细胞生长因子（bFGF的生存），胰岛素样生长因子1（IGF - 1）9]。 <img alt="图1" src="/files/ftp_upload/3200/3200fig1.jpg" /> 图1。编制胚胎小鼠运动神经元的流量计划。示意图说明了隔离和小鼠胚胎运动神经元的文化，不同的步骤。缩写:SC:脊髓;的HBSS:汉克斯平衡盐溶液;百万:运动神经元。 <img alt="图2" src="/files/ftp_upload/3200/3200fig2.jpg" /> 图2从E12.5小鼠脊髓腰部分隔离答:。E12.5小鼠胚胎。下一步，头部和尾部被删除，身体是放置在背的位置B:在胚胎背的位置。在左侧和脊髓右钳固定在其位置的胚胎体。镊子之一，随后被用于切开皮肤和切下的另一种是使用固定在其位置的胚胎的脊髓 C:隔离E12.5小鼠胚胎的脊髓。脊髓（颈椎，胸椎，腰椎）的部分表示。脑膜脊髓周围和背根神经节的一部分，仍然对他们的重视D:脊髓背侧是纵向削减对中央管打开电子邮件:腹侧脑膜现从夷为平地的脊髓。注:带下，腰部分是显着一小截传真:只有腰椎部分的脊髓，然后采取进一步的程序。 <img alt="图3" src="/files/ftp_upload/3200/3200fig3.jpg" /> 图3从腰脊髓E12.5小鼠胚胎胰岛阳性细胞的测定和隔离答:。胰岛- 1 / 2抗体标签胚胎的腰椎脊髓内运动神经元列（红色箭头）和赫斯特标签原子核内的部分。过的第一个E12.5 lumar脊髓。 E12.5小鼠胚胎被浸泡固定在4％多聚甲醛6 H，磷酸盐缓冲液洗3次，与蔗糖按照标准程序处理。 20微米的冰冻切片与低温恒温器和部分immunhistochemical染色处理，根据标准程序11。随后，部分洁具用Hoechst标记细胞核的本地化。请注意，背根神经节神经元Islet-1/2-positive以及和编制程序不包括从隔离程序组织部分B:胰岛- 1 / 2标记（红色箭头）分离的腰，从E12的脊髓细胞。 5个胚胎，左 - 前和右 - 后外源凝集素为基础的preplating。细胞用Hoechst counterstained可视化所有细胞的细胞核C:Islet-1/2-positive细胞的定量前后的凝集素为基础的preplating，D:。Nkx6.1标记后一天在文化E12.5胚胎运动神经元。 counterstained用Hoechst cisualieze所有核细胞。请注意，90％的所有细胞Nkx6.1积极。缩写:SC:脊髓;百万:运动神经元，背根神经节背根神经节。 <img alt="图4" src="/files/ftp_upload/3200/3200fig4.jpg" /> 图4 E12.5小鼠运动神经元表征A和B。富集分离E12.5小鼠腰椎脊髓运动神经元表达p75的正常贸易关系在0天（0div） 在体外 ， 在体外 （2div）第2天。 4％多聚甲醛固定细胞，并根据标准程序，随后染p75 的正常贸易关系。使用赫斯特可视化所有核细胞counterstained C:丰富的运动神经元，后5天色情H 和层粘连蛋白在体外（5DIV）作为培养基质和CNTF的染色为β-Ⅲ-微管蛋白的存在。请注意，这些细胞成长长的突起，和显示一个长（支）的过程，以及一个或多个（轴突和树突）短流程典型的运动神经元的形态。缩写:MN:运动神经元;格: 在体外的日子。 <table border="1"><tbody><tr><td rowspan="2"></td><td colspan="3"> 经过解离而不凝集素为基础的 preplating </td></tr><tr><td> 细胞/ SC的总数 </td><td> 台盼蓝染色阳性细胞[％] </td><td> Islet-1/2-阳性细胞[％] </td></tr><tr><td> 1 </td><td> 995.0 </td><td> 13,1 </td><td> 9,5 </td></tr><tr><td> 2 </td><td> 889.4 </td><td> 8,0 </td><td> 10,0 </td></tr><tr><td> 3 </td><td> 1.180.0 </td><td> 11,0 </td><td> 10,8 </td></tr><tr><td> 平均值 ± SD </td><td> 1.021.0 ± 147.1 </td><td> 10,7 ± 2,6 </td><td> 10,1 ± 0,7 </td></tr><tr><td colspan="4"></td></tr><tr><td rowspan="2"></td><td> 随着外源凝集素为基础的preplating </td></tr><tr><td> 后，细胞数量preplating / SC </td><td> 台盼蓝染色阳性细胞[％] </td><td> Islet-1/2-阳性细胞[％] </td></tr><tr><td> 1 </td><td> 189.6 </td><td> 9,9 </td><td> 70,5 </td></tr><tr><td> 2 </td><td> 168.8 </td><td> 3,7 </td><td> 76,1 </td></tr><tr><td> 3 </td><td> 125.0 </td><td> 0,0 </td><td> 72,5 </td></tr><tr><td> 平均值 ± SD </td><td> 161.1 ± 33.0 </td><td> 4,5 ± 5,0 </td><td> 73,0 ± 2,8 </td></tr></tbody></table> 表1。来自3个不同的隔离程序， 特别是为此的阶段E12.5小鼠胚胎运动神经元代表的隔离程序的摘要。结果总人数或百分比数字± SD。简称:SC:脊髓; SD:标准差。 <table border="1"><tbody><tr><td> 胰岛- 1 / 2阳性细胞与p75的平移[％] </td><td> 胰岛- 1 / 2阳性细胞凝集素为基础的平移[％] </td></tr><tr><td> 92,0 ± 3,5 1 </td><td> 73,0 ± 2,8 </td></tr></tbody></table> 表2。凝集素为基础，基于P75 - 1平移运动神经元细胞数量的比较 。结果给出的百分比数字± SD 。

<ol>
	<li>Vallstedt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+levels+of+repressor+activity+assign+redundant+and+specific+roles+to+Nkx6+genes+in+motor+neuron+and+interneuron+specification.">Different levels of repressor activity assign redundant and specific roles to Nkx6 genes in motor neuron and interneuron specification.</a> Neuron. 31, 743-755 (2001).</li><li>Tsuchida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topographic+organization+of+embryonic+motor+neurons+defined+by+expression+of+LIM+homeobox+genes.">Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes.</a> Cell. 79, 957-970 (1994).</li><li>Lin, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionally+related+motor+neuron+pool+and+muscle+sensory+afferent+subtypes+defined+by+coordinate+ETS+gene+expression.">Functionally related motor neuron pool and muscle sensory afferent subtypes defined by coordinate ETS gene expression.</a> Cell. 95, 393-407 (1998).</li><li>Wiese, S., Beck, M., Karch, C., Sendtner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling+mechanisms+for+survival+of+lesioned+motoneurons.">Signalling mechanisms for survival of lesioned motoneurons.</a> Acta Neurochir. , 21-35 (2004).</li><li>Gavins, F. N., Chatterjee, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+for+the+study+of+mouse+microcirculation+in+anti-inflammatory+drug+research:+focus+on+the+mesentery+and+cremaster+preparations.">Intravital microscopy for the study of mouse microcirculation in anti-inflammatory drug research: focus on the mesentery and cremaster preparations.</a> J. Pharmacol. Toxicol. Methods. 49, 1-14 (2004).</li><li>Bullen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+imaging+techniques+for+drug+discovery.">Microscopic imaging techniques for drug discovery.</a> Nat. Rev. Drug Discov. 7, 54-67 (2008).</li><li>Hazelwood, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entering+the+Portal:+Understanding+the+Digital+Image+Recorded+Through+a+Microscope.">Entering the Portal: Understanding the Digital Image Recorded Through a Microscope.</a> Imaging Cellular and Molecular Biological Functions. , 3-43 (2007).</li><li>Hickey, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-selectin+facilitates+emigration+and+extravascular+locomotion+of+leukocytes+during+acute+inflammatory+responses+in+vivo.">L-selectin facilitates emigration and extravascular locomotion of leukocytes during acute inflammatory responses in vivo.</a> J. Immunol. 165, 7164-7170 (2000).</li><li>Cara, D. C., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+as+a+tool+for+studying+recruitment+and+chemotaxis.">Intravital microscopy as a tool for studying recruitment and chemotaxis.</a> Methods Mol. Biol. 239, 123-132 (2004).</li><li>Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LSP1+is+an+endothelial+gatekeeper+of+leukocyte+transendothelial+migration.">LSP1 is an endothelial gatekeeper of leukocyte transendothelial migration.</a> J. Exp. Med. 201, 409-418 (2005).</li><li>Heit, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+accelerates,+but+is+not+required+for,+neutrophil+chemotaxis+to+fMLP.">PI3K accelerates, but is not required for, neutrophil chemotaxis to fMLP.</a> J. Cell Sci. 121, 205-214 (2008).</li></ol>

没有利益冲突的声明。

基于此凝集素preplating技术的优点是，它是低于p75 的正常贸易关系为基础的平移过程昂贵，和外源凝集素是超过抗体稳定。浓缩列图。 2和标签。 1显示了该程序允许细胞相似的净化和大多数这些细胞表达了运动神经元标记胰岛- 1 / 2。最关键的一步是腰椎脊髓的分离过程。删除的脑膜和背根节（图2）以下的外源凝集素为基础的preplating纯化过程是必不可少的。如果这一直是正确的管理，几乎所有的细胞表达p75的正常贸易关系 （有代表性的图片，请参见图3a ）。胰岛- 1 / 2和p75的正常贸易关系是最有可能的表达之间的区别的原因是因为腰的运动神经元的差异表达上级与下级的胰岛- 1 / 2 10。在胰岛- 1 / 2的表达水平低的情况下，这有可能逃脱immuncytochemical染色和随后的计算中我们的注意力，因为我们是积极与消极的细胞（表1）严格。此外，表1清楚地表明，分离的细胞在一个健康的条件下生存的过程，因为只有几台盼蓝阳性细胞，表明细胞是不可逆的损坏。这种变质的过程，也允许从单一胚胎及胚胎小鼠的混合基因型垃圾因此运动神经元的隔离。总之，这与正常贸易关系的P75抗体6平移过程的替代也有类似的的，如果不是在可能的应用范围相同的能力，提供了一种廉价而高效的的小鼠胚胎运动神经元替代的提纯方法。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>聚- DL -鸟氨酸氢溴酸</td><td>西格玛</td><td> P8638 </td><td></td></tr><tr><td>层粘连蛋白</td><td> Invitrogen公司</td><td> 23017-015 </td><td></td></tr><tr><td>的HBSS </td><td> Gibco公司</td><td> 14170 </td><td></td></tr><tr><td>盖玻片</td><td>热</td><td></td><td>直径10或14毫米</td></tr><tr><td>凝集素</td><td>西格玛</td><td> L5142 </td><td></td></tr><tr><td>细胞培养皿</td><td> NUNC </td><td> 150350 </td><td> Nunclon三角洲面</td></tr><tr><td>马血清</td><td> Linaris </td><td> SHD3250ZK </td><td>每一批百万文化测试</td></tr><tr><td> B27的补充</td><td> Gibco公司</td><td> 17504-044 </td><td></td></tr><tr><td> Glutamax </td><td> Gibco公司</td><td> 35050-038 </td><td></td></tr><tr><td>睫状神经营养因子</td><td>西格玛</td><td> N0513 </td><td></td></tr><tr><td>牛血清白蛋白</td><td> Applichem </td><td> A1391 </td><td></td></tr><tr><td> Neurobasal </td><td> Gibco公司</td><td> 21103-041 </td><td></td></tr><tr><td>镊子</td><td></td><td></td><td>不锈钢，尺寸为4或5 </td></tr><tr><td>胰蛋白酶</td><td>沃辛顿</td><td> LS003707 </td><td></td></tr><tr><td>胰酶抑制剂</td><td>西格玛</td><td> T6522 </td><td></td></tr><tr><td>抗胰岛- 1 </td><td> DSHB </td><td> 39.4D5 </td><td>细胞培养上清</td></tr><tr><td>抗- Nkx6.1 </td><td> DSHB </td><td> F55A10 </td><td>细胞培养上清</td></tr><tr><td>抗p75的正常贸易关系 </td><td> Abcam公司</td><td> Ab8874 </td><td></td></tr></tbody></table> 表3。表特定的试剂和仪器。

lectin-based isolation and culture of mouse embryonic motoneurons

脊髓运动神经元对有丝分裂阶段发展，通过早期胚胎神经系统发育和后来生长出来树突和轴突。神经管神经上皮细胞表达Nkx6.1独特的前体细胞对脊髓运动神经元 1 。虽然有丝分裂运动神经元的走向他们的最终位置，并自己组织成列沿脊髓束2,3。超过90％，所有这些差异化和定位的运动神经元表达的转录因子胰岛1 / 2。他们支配四肢的肌肉，以及身体和内脏的。其中，运动神经元表达脑源性神经营养因子（BDNF）和神经营养因子-3（NT - 3），原肌球蛋白相关激酶B和C（的TrkB，TrkC）的高亲和力受体。他们不表达的原肌球蛋白相关激酶A（TrkA的）4。旁边的两个高亲和力受体，运动神经元表达低亲和力神经营养因子受体p75的正常贸易关系 。 p75的正常贸易关系可以相似，但低亲和力结合所有的神经营养因子，神经营养因子比所有的高亲和力受体结合成熟的神经营养因子。在胚胎的脊髓中，p75的正常贸易关系完全是由5脊髓运动神经元表达。这已被用于发展运动神经元的隔离技术，净化绝大多数的细胞周围的细胞 6 。隔离运动神经元特异性抗体对p75的正常贸易关系的外域（平移）的帮助下却变成了一次实验中使用的抗体量高是由于为平移板的大小是一个昂贵的方法。一个更为经济的另一种方法是使用凝集素。已被证明外源凝集素特异性结合p75的正常贸易关系 ，以及7。下面的方法介绍了一种替代技术，使用一个preplating过程，而不是p75的正常贸易关系抗体麦胚凝集素。凝集素是一个非常便宜的替代p75 的正常贸易关系的抗体和净化等级使用外源凝集素与p75的正常贸易关系抗体。可以用这种方法分离出胚胎脊髓运动神经元，生存和发展，突起。

Watch this Scientific Journal Video about Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons at JoVE.com

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

隔离从小鼠胚胎的脊髓运动神经元的另一种方法是描述。该方法考虑外源凝集素，可以绑定到低亲和力神经生长因子受体P75NTR的事实。基于这种外源凝集素preplating允许一个类似与P75NTR对特定抗体的纯化。

基于凝集素的分离和培养的小鼠胚胎运动神经元

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

lectin-based-isolation-and-culture-of-mouse-embryonic-motoneurons

Research

JoVE Journal

Neuroscience

17.9K 次观看.  Ruhr-University Bochum. 隔离从小鼠胚胎的脊髓运动神经元的另一种方法是描述。该方法考虑外源凝集素，可以绑定到低亲和力神经生长因子受体P75NTR的事实。基于这种外源凝集素preplating允许一个类似与P75NTR对特定抗体的纯化。

视频: Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

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

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection和胚胎小鼠海马和皮层神经元的原代培养

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

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 span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

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 ...

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

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

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 Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

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 ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

解剖，文化与分析<em&gt;非洲爪蟾</em&gt;胚胎视网膜组织

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

小鼠脑皮层星形胶质细胞的分离和培养

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

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 ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

分离和成人神经干细胞的培养从鼠标Subcallosal区

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...