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 。

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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

Universidad de Cartagena UDEC

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

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 ...

科研

神经科学

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 ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

一个<em在体外</em从成年小鼠的肌间神经丛制备隔离肠道神经元和神经胶质

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

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 postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

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 ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

高效的小鼠胚胎干细胞分化成运动神经元

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

生物学

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, and spinal muscular atrophy, which are all associated with muscular atrophy. Primary cultures of spinal MNs have been used widely to demonstrate the involvement of specific genes in such diseases and characterize the cellular consequences of their mutations. This protocol models a primary MN culture derived from the seminal work of Henderson and colleagues more than twenty years ago. First, we detail a method of dissecting the anterior horns of the spinal cord from a mouse embryo and isolating the MNs from neighboring cells using a density gradient. Then, we present a new way of efficiently transfecting MNs with expression plasmids using magnetofection. Finally, we illustrate how to fix and immunostain primary MNs. Using neurofilament mutations that cause Charcot-Marie-Tooth disease type 2, this protocol demonstrates a qualitative approach to expressing proteins of interest and studying their involvement in MN growth, maintenance, and survival.

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

The goal of this technique is to prepare a highly enriched culture of primary motoneurons (MNs) from murine spinal cord. To evaluate the consequences of mutations causing MN diseases, we describe here the isolation of these isolated MNs and their transfection by magnetofection.

小鼠原代多元体的体外模拟？

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

胚胎小鼠神经干细胞的分离与培养

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS) when compared to spinal motor neurons (SMNs). The ability to isolate and culture primary mouse CN3s, CN4s, and SMNs would provide an approach to study mechanisms underlying this selective vulnerability. To date, most protocols use heterogeneous cell cultures, which can confound the interpretation of experimental outcomes. To minimize the problems associated with mixed-cell populations, pure cultures are indispensable. Here, the first protocol describes in detail how to efficiently purify and cultivate CN3s/CN4s alongside SMNs counterparts from the same embryos using embryonic day 11.5 (E11.5) IslMN:GFP transgenic mouse embryos. The protocol provides details on the tissue dissection and dissociation, FACS-based cell isolation, and in vitro cultivation of cells from CN3/CN4 and SMN nuclei. This protocol adds a novel in vitro CN3/CN4 culture system to existing protocols and simultaneously provides a pure species- and age-matched SMN culture for comparison. Analyses focusing on the morphological, cellular, molecular, and electrophysiological characteristics of motor neurons are feasible in this culture system. This protocol will enable research into the mechanisms that define motor neuron development, selective vulnerability, and disease.

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons.

从产前Islmn:GFP转基因小鼠的奥库罗运动、特罗赫利尔和脊柱运动神经元的分离与培养

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral ...

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.

Axonal transport is a crucial mechanism for motor neuron health. In this protocol we provide a detailed method for tracking the axonal transport of acidic compartments and mitochondria in motor neuron axons using microfluidic chambers.

使用微流体腔系统在运动神经元培养物中有机细胞的斧头迁移

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...

Isolation and Culturing of Cells from Mouse Embryo Dorsolateral Telencephalons

Source: Landeira, B. S., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/56063">Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System.</a> J. Vis. Exp. (2017)
This video demonstrates a protocol for isolating and culturing primary cerebral cortex cells from the dorsolateral telencephalon of mouse embryos.

小鼠胚胎背外侧端脑细胞的分离和培养

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...