我们特别感谢利诺金，史蒂夫古兹曼和Ujit的Satyarthi的，这种方法在开发过程中的技术援助。迈伦·霍桑，理查德·Spengel，罗伯托·罗哈斯加工室使用，提供了必要的技术援助。值得一提的是，罗伯托·罗哈斯图4。我们也感谢斯科特·弗雷泽发展的趋化试验前的宝贵意见。这项工作得到部分支持，NIH-MBRS分数5S06GM048680-13 MEDB和加州州立大学北岭分校研究生论文支持计划奖CW。

1。第1天:过夜培养于盖玻片上的躯干神经管分离<ol><li> 56小时的孵育小鸡的鸡蛋在38℃中取出的鸡蛋孵化，轻度喷用70％的乙醇，然后让他们干。将鸡蛋打碎成紫外线消毒的玻璃盘。 </li><li>从每个胚胎中提取蛋黄，将其放置在小鸡林格氏。通过第一弯剪刀切割周围血岛，然后，用生硬的镊子，挑胚胎的胚外膜，并将其放置在无菌塑料培养皿中含有小鸡林格氏液。 </li><li>隔离每个胚胎的树干修剪多余的胚外膜以及颅，迷走神经，骶轴向水平，使用钨针（图1）。首先，选择约9胚胎阶段之间HH15-17 11。对于阶段HH15和，前脑和后脑轴形成一个锐角，因此出现的头部倾斜尾端。在阶段HH17，尾芽和倾斜腹部，但不包含体节。有了一个钨针，修剪掉至约2毫米的胚外膜从胚胎和切断前的体节10任何胚胎组织。同时删除所有的尾部的胚胎组织从周围的第五个新形成的体节。 </li><li>将孤立胚胎中继线在中性蛋白酶（0.24 U / ml的DMEM培养基）孵育1小时15分钟，在37℃和5％CO 2的 。树干一旦开始孵化，开始准备6的盖玻片（CS）培养NT植（步骤1.5-1.8）。 </li><li>冲洗6在70％乙醇中的CS（在无菌水，超纯水稀释），然后允许它们干燥。使用实验室的标记，画一个圆圈，在每个CS的中心是直径在1厘米左右（这个圈子以后可以帮助您确定已应用于纤维连接蛋白涂层）。在同一个面上的每一个CS，写的字"是"（或其他一些非对称的字或形状）绘制的圆外（这将有助于ŸOU识别是否正面临着显着的CS向上或向下）。 </li><li>每个CS放置在一个单独的40×10毫米的无菌培养皿与标记的表面朝下，并允许菜坐杀菌10分钟的UV灯的下方开放。 </li><li>应用60微升纤连蛋白（FN，10微克/毫升的DMEM）的CS未标记的表面涂覆，同时确保在整个区域内的1厘米的圆。将菜在37°C孵育30分钟，然后小心吸纤维连接蛋白，每一个CS。 </li><li>加入250微升"文化"培养基[含L-谷氨酰胺（2毫摩尔），青霉素（100U/ml），链霉素（100微克/毫升），和8％胎牛血清（FBS）的] FN涂覆面积CS。将含有每个CS的菜肴，在37℃和5％CO 2中 ，直到已被隔离的NT。 </li><li>把所有的孵化胚胎干一个5公分的玻璃培养皿中L15介质，并开始解剖每个NT，用细镊子和钨针（图1）。 Çarefully片沿边境NT和体节用锋利的钨针，同时注意不要损坏NT。它往往是更容易，开始隔离每个NT的尾部，最末端的主干。 </li><li>选择直最长的NTS培养过夜（NTS大约8和15体节间长，建议）6。引培养基用微尖，转让的6 NTS自己事先准备好的CS（从1.5到1.8）。要确保NT不保持浮在表面。如果NT是浮动的，滴介质上，直到它下沉用微。 </li><li>将每道菜在37°C，5％CO 2晚 。要小心，以确保每一个NT是在其各自的CS FN-涂布面积紧接的菜放置在孵化器中（通过使用作为参考绘制圆的步骤1.5）。更好地调整每个NT的位置，如果需要的话，可用于微量。 </li><li>地点在至少2 毫升培养基（无血清）到一个无菌的15毫升离心管中，并在37℃和5％CO 2中孵育过夜。发布帽稍微拧松，以允许介质的pH调整过夜。预孵化介质有助于防止泡沫的形成，在你的房间，这可能干扰的分子梯度的建立是非常重要的。这样的"预孵育"中，应采用未来所有的步骤。在没有使用时，此媒体应孵育在37℃下</li></ol> 2。第2天:载入的改性Zigmond室和延时分析细胞迁移<ol><li>出的6 NTS培养，选择最适合分析3文化。一般来说，NCC文化，应选择有至少一个长的，直边（图2A）。最好的文化将被用于加载和电影3的改性Zigmond室在一天，每一个不同的治疗。在3文化，选择一个为LOADIN克的第一腔室和返回他人培养箱购买使用。 </li><li>用棉签，适用于薄，甚至一层凡士林周围的水库，桥梁，一个被修改的Zigmond室。 </li><li>有了一个钨针，轻轻地移除NT从CS，而离开周边连接到的CS表面的净捐助国。 ，用钢笔记住的方向直的边缘的NCC文化的标记的菜。 </li><li>将几滴预孵育介质上桥。拿起CS用细镊子，轻拍边缘的CS反对一个的Kimwipe以去除大部分的旧培养基 ，然后立即将CS修改后的Zigmond室，使被拍摄下来的文化中心，直边的长度的桥梁和大致垂直的桥型储层边界（图2A，B）。 </li><li>使用倒置显微镜，直NCC边界移动到上侧的最近的第桥Ë水库将包含可疑的趋化（图2B的控件，这将对应的水库加载第二）。此外，更精细地对齐的文化的直边是垂直的桥型储层边界。 </li><li>仔细，但牢固地按CS到凡士林本上Zigmond室，确保完全密封的腔室，然后放置额外的凡士林，沿边缘的CS，以进一步确保这将是气密。精细调整角度的NCC边界再次校正在密封过程中的任何移动。 </li><li>装入的水库，将不包含疑似趋化第一（图2B）。这样做的装载1毫升注射器（25 G连接的针x 1.5英寸），约300微升的预孵育介质和注入介质，额满为止（入水库同时小心不产生任何气泡在水库）。将水库两侧与SUFFicient量的凡士林之前加载下一个水库。 </li><li>重复步骤2.7，使用预培养的培养基中的候选人趋化只是这一次。这是关键的时候产生的分子梯度的文化，始终加载水库进行测试后加载水库缺乏测试分子的分子。 </li><li>将加载的Zigmond厅在37°C孵育1小时，拍摄前。图片直边界的的NCC文化，在90秒的时间间隔3小时，而培养在大约37°C（图2A，B）。创建任何电影之前，一定要调整，使相机获得的图像边缘对准并接触边缘的桥梁接壤的最后一个水库加载（图2B，上面板;虚线框表示理想成像位置）。这将有利于以后的软件通过规范的分子梯度和日的方向性的分析生产Ë距离水库拍摄的每部电影。 </li><li>对于控件，重复步骤2.2-2.9为每个其他两个NCC文化选择（在步骤2.1），但用适当的培养基中，填充每个水库。对于一种类型的控制与预培养介质不含有要测试的分子填补两个水库。对于第二个控制治疗，首要的桥梁与预培养的培养基中可疑的趋化之前安装的CS几滴。然后，加载都用相同的培养基中怀疑趋化水库。 </li><li>使用ImageJ的（NIH） 手动跟踪 <a href="http://rsb.info.nih.gov/ij/plugins/track/track.html">（rsb.info.nih.gov / IJ /插件/曲目/ track.html）的</a>和趋化和迁移工具V1.01 <a href="http://www.ibidi.de/applications/ap_chemo.html">（www.ibidi.de /应用/ ap_chemo.html的）</a>插件来追踪周边NCC的迁移沿直线边界的文化Ĵ乌斯季成像和分析各种参数的迁徙轨迹（图2B-C）。 </li></ol> 3。代表性的成果: 示出的轨迹从电影许多中继线NCC的响应于使用上述技术的候选趋化的细胞的样品（图2D）。在这个例子中的大部分细胞的积极响应，显示了趋化因子梯度净迁移人数（显示为红色）。轨迹数据可用于分析细胞迁移以及其他属性。 为了在视觉上评价在改性Zigmond室，IgM抗体共轭的Alexa Fluor 488（分子量〜900 kDa的）施加的梯度被加载到所述第二储改性Zigmond室（在大约40μg/ ml的H 2 O）。渐变1小时和26小时后还是有点，但大大减少了50小时（图3）。如果分子进行测试，然后应用梯度无线会降低速度比显示。 <img alt="图1" src="/files/ftp_upload/3330/3330fig1.jpg" /> 图1。外植干线新界南总纤连蛋白包被盖玻片上培养过夜 。由于主干NCC的残缺不全的背NT位于相邻的体节8月28日，该段的NT显微切割分离和培养过夜，纤维连接蛋白涂层CS允许的NCC的从NT植的移民。 8-15体节间长，比较直的隔离NTS最适合过夜培养，因为他们往往​​会产生NCC文化与较长的直边界。神经管的引起其他神经嵴轴向水平的地区在一个较小的字体显示。 ，体节。 <img alt="图2" src="/files/ftp_upload/3330/3330fig2.jpg" /> 图2迁移取出的主干NCC的评估方法。使用一个的改性Zigmond室。 （A）准备拉长躯干NCC文化至少有一个长而直的边界过夜培养的NTS和由此产生的NCC文化的选择实验。选定文化的最长的直边界，然后垂直于定位的桥型储层边界，因此，平行于未来应用梯度矢量。（B）的经过精细调整的位置上Zigmond室和密封的NCC文化盖玻片室，该室被加载。当测试趋化，水库，将不包含可疑的趋化（ - ）第一个被加载和密封。然后，其他的水库被加载与怀疑趋化（+）和密封。周边NCC的沿着先前选定的边界进行成像和跟踪使用手动跟踪插件ImageJ的（下图）。（C）许多候鸟的响应特性与所施加的梯度可以评估的基础上跟踪数据。比如，一个趋化指数可以衍生除以细胞的位移沿x轴的总距离它已迁移，（D）的一个有吸引力的响应的一个例子被示出由一个细胞轨迹情节最初产生的趋化性和迁移为ImageJ的工具插件。每条轨迹的开始点被设置为原点（0,0）。注意要多细胞转向的的趋化source.The中心的所有单元格的质量在最后的位置（蓝十字相同的权重，所有的细胞）也接近单核细胞趋化源。 NCC的，神经嵴细胞;红色轨道，细胞迁移向容器装有一个怀疑的单核细胞趋化黑色轨道，细胞迁移距​​离（+），更高的趋化浓度（ - ），低级的趋化浓度。 <img alt="图3" src="/files/ftp_upload/3330/3330fig3.jpg" /> <stron克&gt;图3。 强度跨桥公司的Alexa Fluor 488 IgM抗体共轭物的添加后，在不同的时间改性Zigmond室室中装入在协议中所描述的类似的方法，与主要的例外，预培养水（而不是预培养介质）被用来抗体稀释至40微克/毫升，在桥的端部（远离片采取上述强度分布）和小的空气袋本。最初，没有梯度是目前大部分的桥梁。按1公顷梯度成立，并维持到26小时。 50小时的梯度的存在，是在不同地区的桥梁不一致，存在时，陡峭的梯度大大减少。穿过大桥（一桥水库边界），使用相同的片A​​xioVision 4.6软件产生的所有配置文件。请注意，即使在空气口袋里目前，梯度不中断。高，高强度，低，强度低; x轴，整个桥（2毫米）的宽度的距离（+），储层装有的Alexa Fluor 488 IgM抗体共轭（ - ），储集层的共轭不加载。 <img alt="图4" src="/files/ftp_upload/3330/3330fig4.jpg" /> 图4。改性Zigmond室规格所示是示这里使用随着其尺寸规格（±0.2毫米）的改性Zigmond室。测量可以适度调整，以配合个人喜好。 补充协议:制作改进的Zigmond厅请参阅图4作为参考下面的协议: <ol><li>购买一张3/16"厚的抛光亚克力（4.45 mm实际厚度）。 </li><li>使用一台锯，切割室空白过大的粗糙的尺寸33.25毫米x64.57毫米的。这允许3.175毫米的额外的用于加工的材料。 </li><li>将用老虎钳室空白。有了一个铣床和6.35毫米（1/4"）的端铣刀位，精加工的腔室的侧面到其精确的尺寸:30.07毫米x61.39毫米。 </li><li>腔室定位空白的铣床上并定位沿x和y轴与一个边器的中心的坯件，然后零的中心位置。 </li><li>采集腔室高度（z轴）的顶表面接触的端铣刀位和零的高度。 </li><li>使用3.91毫米（0.154"）的端铣刀位，偏移位3.03毫米沿x轴（正方向）为第一储。开始加工成2.84毫米的深度的腔室移动的同时沿y-轴（正方向）至7.62毫米（0.300"），然后遍历到7.62毫米（0.300"）在相反的方向（负）的一个完整的水库长度为15.24毫米（0.600"）。抵销位至3.03毫米（0.119"）沿x轴的（负方向），并重复相同的过程的第二水库。 </li><li>室的位置在它的边缘，钻一个孔，用1.09毫米（0.043英寸）钻头（共4个），每个水库的水库在实验过程中加载介质腔侧连接端。 </li><li>在温暖的肥皂水中浸泡室，以帮助消除任何化学污染物。 </li><li>浸泡和冲洗腔室，以及在双蒸水中，以消除任何肥皂。该商会是现在已经准备好使用上述。 </li></ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+direct-viewing+chemotaxis+chamber.">A new direct-viewing chemotaxis chamber.</a> Journal of Cell Science. 99, 769-775 (1991).</li></ol>

没有利益冲突的声明。

在主干NCC的趋化研究已经证明了具有挑战性的一系列原因。主干的NCC的构成一个异构干细胞的人口将分化，如果培养长期的，因此，躯干NCC的，必须从主外植的中继级NT。传统的方法来研究均匀分布在体外的细胞群的趋化反应是难以在主干NCC的测试，因为他们首先需要的细胞是孤立的均匀再接种的趋室（例如，Boyden小室12）。为外植几十个NTS可能会被要求创建一个均匀分布的主干NCC的，足够的，甚至一个实验隔离足够NCC的可能是很乏味的。此外，NCC的都没有抬，混合，均匀重新分配他们的自然背景。因此，NCC的行为可能以执行某些常规趋化实验，再接种完全不同的在体内条件在迁徙行为方面。 在这里，我们描述一个技术评估主干NCC趋化以一种更自然的情况下。一个主要的挑战进行评估时，没有一个均匀分布的趋化反应的主干NCC的是NCC的内原有的外植体培养，除了他们天生的异类身份的强烈固有的极性。例如，躯干NCC的倾向主要是垂直迁移（客场）从NT时，在文化13。的各种影响因素，树干NCC内的外植体培养的极性可以如此强大，它是很难最后确定的趋化梯度诱导细胞极性的变化。使用我们的新的修改后的Zigmond分析，它是可以区分天然细胞极性固有的外植体的文化和趋化梯度的变化，同时也监控其他候鸟参数的<html> TERS，如持久性和速度。我们观察到，每个边界的的外植主干NCC文化所面临的角在很大程度上决定着固有的方向性的领先优势，边境主干NCC的。举例来说，如果一个边界的NCC文化朝北，然后NCC的从该边界的平均方向性偏北。修改后的分析，最大限度地减少变异所造成的固有的方向性的领先优势NCC的规范NCC边境，从他们的角度迁移的趋化梯度（图2A）。通过相对较直的主干NCC文化边界，总是选择最先进的NCC的定位，直的NCC边界垂直的梯度，然后只跟踪，边境上的领先优势细胞的迁移，一种是能够探测到的变化在净捐助国朝向或远离从趋化源，否则将去未被发现躯干的平均方向性。 ENT"&gt;下面是来自上述协议的诸多好处的总结: <ol><li>它提供了追踪活细胞迁移的能力。 </li><li>的改性Zigmond室可以制作一个相对定义的梯度，可以可视化与荧光分子（图3）。 </li><li> Zigmond室避免一定的局限性，Boyden小法先前所描述的其他10,14的标准。 </li><li>我们的的修改Zigmond室可以在一个非常低的成本（材料，估计在不到1美元可重复使用的室）。 </li><li>由于神经管的盖玻片上培养，并购买用凡士林密封，可以容易地选择的文化的取向相对于未来的应用梯度。 </li><li>胰蛋白酶消化分离或细胞刮不是必需的。 </li><li>只有少量的NT隔离是必要的。 </li><li> NCC文化是类似的分布时相比，细胞在体内发现的树干NCC的均匀的分布被提升，并再接种。 </li></ol>这些优点也可能适用于类似的其他类型的细胞的初级外植体培养物，因此，这种方法可以有更广泛的用于评价其他取出的干细胞的自然洄游行为可取。 虽然改性Zigmond室法的优点是很多的，它也有一些局限性。的的标准Zigmond腔的一些缺点已经讨论过由过去的研究14。我们修改的Zigmond测定的限制之一是用于安装在CS和堵塞每个水库孔使用凡士林。凡士林允许灵活精确地定位一个NCC相的趋化梯度的文化，但导致在CS和桥，它可能导致所施加的梯度14变异之间的距离的可变性。此外，安装CS和密封腔室与凡士林经常陷阱的空气在腔室的小口袋，这可能导致桥14的流过少量压缩。这种流动可能会在一些实验和破坏分子梯度，导致变异增加的结果。虽然这是可能的情况下，没有观察到严重的梯度中断时，可视化使用的Alexa Fluor 488 IgM抗体共轭物（图3），即使在空气口袋本朝向桥的端部（离测定荧光强度的区域） 。因此，即使可能有潜在的破坏的Alexa Fluor 488 IgM抗体梯度的条件下，将梯度不损害面积测量（更接近中间的桥梁）的桥梁。如果一个需要长期的梯度，是高度稳定的和可预测的，那么该试验中可能会不足够。然而，这样的精确往往不需要，相比其他一些常见的趋化实验（如Boyden小实验或某些实验使用趋化浸泡过的珠），梯度可能是更稳定的和可预测的。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> DMEM </td><td>欧米茄科学</td><td> DM-22 </td><td></td></tr><tr><td>青霉素链霉素解决方案中</td><td>欧米茄科学</td><td> PS-20 </td><td> 100X股权集中度</td></tr><tr><td> L-谷氨酰胺</td><td>欧米茄科学</td><td> GS-60 </td><td> 100X股权集中度</td></tr><tr><td>胎牛血清</td><td>欧米茄科学</td><td> FB-11 </td><td>地段＃105247（或其他相媲美） </td></tr><tr><td>修改Zigmond室</td><td>自制</td><td> N / A </td><td>水库容积:160微升EA;其他规格，请参阅图。 4，补充制造协议</td></tr><tr><td>细胞培养皿</td><tD&gt;丹维尔</td><td> T6040 </td><td> 40×10毫米</td></tr><tr><td>纤维连接蛋白</td><td> BD </td><td> 354008 </td><td> 10倍的股票准备在1毫升H 2 O和9毫升DMEM稀释1毫克FN </td></tr><tr><td>盖玻片</td><td>费舍尔</td><td> 12-548-B </td><td>预清洗; 22 x 22毫米</td></tr><tr><td> L15介质</td><td> Thermo Scientific的</td><td> SH30525.02 </td><td></td></tr><tr><td>凡士林</td><td>舒适</td><td> 011110794642 </td><td> 100％ </td></tr><tr><td>离心管</td><td> Biologix </td><td> 10-9152 </td><td> 15毫升</td></tr><tr><td>中性蛋白酶</td><td>电池系统</td><td> 4Z0-850 </td><td> 10X股权集中度</td></tr><tr><td>注射器</td><td> BD </td><td> 309602 </td><td> 1毫升</td></tr><tr><td>针</td><td> BD </td><td> 305127 </td><td> 25 G x 1.5英寸</td></tr><tr><td>的Alexa Fluor 488-IgM阳性</td><td>我nvitrogen </td><td> A21042 </td><td>股票是2毫克/毫升; 7摩尔的染料/摩尔IgM抗体</td></tr><tr><td>解剖镊</td><td> FST </td><td>杂项。 </td><td>杜蒙特＃5或55;直嘴;不锈钢或钛</td></tr><tr><td>钨针</td><td> N / A </td><td> N / A </td><td>首页放置在针座</td></tr><tr><td>钝钳</td><td>蒂曼</td><td> 160-18 </td><td>用于转移胚胎林格从蛋黄中的</td></tr></tbody></table>补充协议:制作改进的Zigmond厅请参阅图4作为参考下面的协议: <ol><li>购买一张3/16"厚的抛光亚克力（4.45 mm实际厚度）。 </li><li>使用一台锯，切割室，以粗糙的尺寸33.25毫米x64.57毫米中的空白过大的。这允许3.175毫米的额外的用于加工的材料。 </li><li>在六室设置空白本身。有了一个铣床和6.35毫米（1/4"）的端铣刀位，精加工的腔室的侧面到其精确的尺寸:30.07毫米x61.39毫米。 </li><li>腔室定位空白的铣床上并定位沿x和y轴与一个边器的中心的坯件，然后零的中心位置。 </li><li>采集腔室高度（z轴）的顶表面接触的端铣刀位和零的高度。 </li><li>使用3.91毫米（0.154"）的端铣刀位，偏移位3.03毫米沿x轴（正方向）为第一储室开始加工成2.84毫米的深度，同时沿y轴移动（正方向）至7.62毫米（0.300"），然后遍历至7.62毫米（0.300"），在相反的方向（负）的长度为15.24毫米（0.600"）的一个完整的水库。偏移比特沿x轴方向（负方向）至3.03毫米（0.119"），并重复同样的过程，所述第二储。 </li><li>在它的边缘放置室钻一个孔，用1.09毫米（0.043英寸）钻头（共4个），每个水库的水库在实验过程中加载介质腔侧连接端。 </li><li>在温暖的肥皂水中浸泡室，以帮助消除任何化学污染物。 </li><li>浸泡和冲洗腔室，以及在双蒸水中，以消除任何肥皂。该商会是现在已经准备好使用上述。 </li></ol>

analysis of trunk neural crest cell migration using a modified zigmond chamber assay

神经嵴细胞（NCCS）是一个流动人口的细胞存在于脊椎动物的发展，移民从背神经管（NT）后发生上皮-间质转化1,2。在EMT，NCC的长距离迁移，沿刻板的途径，直到他们达到他们的目标。 NCC的区分成为一个巨大的数组类型的细胞，包括神经元，神经胶质细胞，黑素细胞和嗜铬细胞1-3。 NCC的接触和认识到自己的正确的目标位置的能力是基本适当的主干NCC-源性成分结构的形成。因此，阐明机制的指导主干NCC迁移的问题具有十分重要的意义。大量的分子已被证明来指导NCC迁移4。例如，躯干NCC的Semaphorin，和ephrin和缝配体的5-8负指导线索，如被击退。然而，并非直到最近，已确定9的任何趋化因子的主干NCC的。 常规的体外方法研究的贴壁细胞的趋化行为的工作最好与永生化的，均匀分布的细胞，但更具挑战性的适用于某些初级最初缺乏均匀的分布和迅速分化（如NCC的）的干细胞培养。均匀化分布的主干NCC的趋化研究的一种方法是从小学NT植文化的主干NCC的隔离，然后抬起replate他们几乎100％汇合。然而，这种电镀的方法需要大量的时间和精力，到取出足够的细胞，是残酷的，分布躯干的NCC的在一个不同的方式，发现在体内条件。 在这里，我们报道了在体外的方法，是可以评估的趋化和其他迁徙的主干NCC的回应没有requirin嘎同质细胞分布。此技术利用延时成像伯，泰然自若改性Zigmond室（一个标准Zigmond室其他地方描述10）内的中继线净捐助国。通过暴露在培养的周边的中继线净捐助国到趋化梯度是垂直于他们的预测的自然方向性，由所施加的趋化梯度诱导洄游极性的改变可以被检测。这种技术是廉价的，需要只有两个NT外植体每重复处理的培养，避免恶劣的细胞起重（如胰蛋白酶消化），叶中继线NCC的在一个更相似的分布，在体内条件下，外植和实验之间的时间量减少（这可能会降低分化的风险），并允许时间的推移众多的候鸟特性的评价。

Watch this Scientific Journal Video about Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay at JoVE.com

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

取出的细胞迁移（躯干神经嵴细胞）的方法来分析。这种方法是廉价的，温柔的，和能够区分趋化学增活现象和其他因素的影响，如在主躯干神经嵴细胞培养的细胞 - 细胞相互作用所产生的迁徙极性的。

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 ...

analysis-trunk-neural-crest-cell-migration-using-modified-zigmond

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.4K 次观看.  California State University, Northridge. 取出的细胞迁移（躯干神经嵴细胞）的方法来分析。这种方法是廉价的，温柔的，和能够区分趋化学增活现象和其他因素的影响，如在主躯干神经嵴细胞培养的细胞 - 细胞相互作用所产生的迁徙极性的。

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

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

成年鼠神经干细胞的分离和扩大使用神经干含量

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

科研

神经科学

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 ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

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). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

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). ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

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

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

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

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 the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

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 ...

Co-analysis of Brain Structure and Function using fMRI and Diffusion-weighted Imaging

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when studying the brain, as the functional role that a brain area fulfills may be largely defined by its connections to other brain areas. In this report, we describe a novel, non-invasive approach for relating brain structure and function using magnetic resonance imaging (MRI). This approach, a combination of structural imaging of long-range fiber connections and functional imaging data, is illustrated in two distinct cognitive domains, visual attention and face perception. Structural imaging is performed with diffusion-weighted imaging (DWI) and fiber tractography, which track the diffusion of water molecules along white-matter fiber tracts in the brain (Figure 1). By visualizing these fiber tracts, we are able to investigate the long-range connective architecture of the brain. The results compare favorably with one of the most widely-used techniques in DWI, diffusion tensor imaging (DTI). DTI is unable to resolve complex configurations of fiber tracts, limiting its utility for constructing detailed, anatomically-informed models of brain function. In contrast, our analyses reproduce known neuroanatomy with precision and accuracy. This advantage is partly due to data acquisition procedures: while many DTI protocols measure diffusion in a small number of directions (e.g., 6 or 12), we employ a diffusion spectrum imaging (DSI)1, 2 protocol which assesses diffusion in 257 directions and at a range of magnetic gradient strengths. Moreover, DSI data allow us to use more sophisticated methods for reconstructing acquired data. In two experiments (visual attention and face perception), tractography reveals that co-active areas of the human brain are anatomically connected, supporting extant hypotheses that they form functional networks. DWI allows us to create a "circuit diagram" and reproduce it on an individual-subject basis, for the purpose of monitoring task-relevant brain activity in networks of interest.

We describe a novel approach for simultaneous analysis of brain function and structure using magnetic resonance imaging (MRI). We assess brain structure with high-resolution diffusion-weighted imaging and white-matter fiber tractography. Unlike standard structural MRI, these techniques allow us to directly relate anatomical connectivity to functional properties of brain networks.

使用功能磁共振成像和弥散加权成像脑结构与功能分析

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when ...

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 ...

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

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 research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

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 ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

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 ...