Our research program is funded through the Emmy Noether-Program of the German Research Foundation (DFG), grant PR1132/3-1. Further support by the M&#252;ller-Fahnenberg Foundation of the University of Freiburg is gratefully acknowledged. This study was supported in part by the Excellence Initiative of the German Research Foundation (GSC-4, Spemann Graduate School).

1.神经细胞收获<ol><li>评估通过显微镜: <ol><li>在此之前启动一个实验，请与明场或相衬显微镜文化的地位。 注意:尽管从解剖得到初级神经组织是，在原则上，同样适合于流式细胞仪分析14,28，请注意，该协议的重点是从体外神经细胞系统获得的细胞。 </li></ol></li><li>收获细胞7: <ol><li>轻轻洗涤贴壁细胞用Mg 2+的盘/烧瓶/ 钙自由磷酸盐缓冲的盐水（PBS）在室温下（ 如 5毫升的T75烧瓶中，每井3毫升6孔板或10毫升10cm皿）。 注:PBS用于整个协议是Mg 2+ / 钙免费的。 <ol><li>用于视频例如，使用的SH-SY5Y成神经细胞瘤细胞的T75烧瓶中，在80％的汇合。适用的情况下的附加的洗涤步骤大量的碎片存在于盘中。 </li><li>考虑洗血清PBS含48白蛋白，珀，或聚蔗糖49梯度离心和/或市售珠50,51。去除髓鞘和其它脂质或其他污染物是至关重要的，特别是当正在使用成人原发性组织来源。 </li></ol></li><li>添加预热（37℃）胰蛋白酶替换在覆盖组织培养皿的整个表面上适当的音量。 注:另外，考虑的Accutase或其他酶消化选项。这关键的一步，可能表面抗原的表达产生负面影响（见7）。 </li><li>孵化菜/烧瓶，在37℃进行2 - 5分钟（取决于细胞类型），以使细胞分离。轻轻拍打组织培养皿或冲洗用血清学吸管打跑的细胞。避免过度消化（因为这可能会导致电池损耗和凝固在后面的步骤）。 </li><li> Quench胰蛋白酶置换，加入两倍的流动缓冲液（在PBS中的2％FBS）的体积和收集细胞在15ml锥形管中。 </li><li>轻轻磨碎使用微升吸管将细胞悬浮液（100 - 1,000微升）或5ml的血清吸管以制备单细胞悬浮液。 </li><li>离心细胞，在220×g离心5分钟，在25℃下。小心吸出上清液留下沉淀的后面。 </li><li>重悬浮沉淀的流动缓冲器的适当体积，取决于粒料（尺寸例如为SH-SY5Y细胞中的一个汇合的T75烧瓶中的典型产率为至少10×10 6个细胞，在这种情况下，将细胞重新悬浮于5毫升的流动缓冲液）。 注意:如果较大的块或凝血观察，筛选过30 - 100微米的网格。 </li></ol></li><li>细胞计数52: <ol><li>在TR的一个体积调小等分的细胞悬浮液到一个离心管中，并稀释在限定的比率ypan蓝色或转移至血细胞计数器或自动细胞计数系统之前的替代生存能力染料。 </li><li>通过加入流动缓冲液或PBS中适当体积用0.1％BSA（如果继续用CFSE标记）稀释细胞悬浮液以1×10 6个活细胞/ ml的浓度。 注:碘化丙锭，7氨基放线菌素D，膜联蛋白V和市售可定影活力检测试剂盒代表，以评估细胞的生存力的替代方案。此外，使用胱天蛋白酶-3的荧光凋亡测定法如先前描述的53可以被使用。这些试剂如果包括所有样品中，可能会限制后续步骤中选择荧光通道将被"占用"。 </li></ol></li></ol> 2.细胞内染料标记用CFSE（图3） <ol><li>稀释CFSE至所需原料浓度可以很容易地使用。 注:对于这些实验股票浓度0.01毫米中。确定的CFSE的最佳工作浓度凭经验。 </li><li>添加10微升每ml细胞的0.01毫CFSE溶液（1.3.2节，在PBS + 0.1％BSA的1×10 6个细胞/ ml的浓度）为0.1微米的最终浓度。简要涡旋拌匀。 注意:这里使用CFSE浓度约10倍比那些在增殖实验常用的降低，因此电池中染料的毒性是最小的。我们没有观察到对细胞活力的不利影响。 </li><li>孵育5分钟，在RT下持续振荡（200转）。避光。 </li><li>通过加入5倍体积的缓冲液流动到管淬灭染料。离心在94×g离心5分钟，在室温。 </li><li>弃去上清液留下沉淀后面。重新悬浮细胞以5倍体积的缓冲液流动。 </li><li>离心在94×g离心5分钟，在室温。弃去上清液并重新悬浮的细胞在流缓冲器以1×10 6个细胞的浓度/<html>毫升。 </li><li>添加相同数量的感兴趣的染色细胞悬浮液未染色细胞。进行表面抗原染色协议（ 第3节 ）。 </li></ol><img alt="图3" src="/files/ftp_upload/52241/52241fig3highres.jpg" width="700" /> 图3.检测通过CFSE染料标记。的SH-SY5Y成神经细胞瘤细 ​​胞两种细胞系之间的差分的CD表面抗原表达的是预先标记相比，未标记的BJ成纤维细胞用CFSE用于随后鉴定。共染色混合样品（右图）与表面标志物CD24或CD54（两者结合到APC）的证明，细胞系很容易分辨由于CFSE染色（箭头= SH-SY5Y;箭头= BJ成纤维细胞）。大多数的SH-SY5Y细胞表达CD24但不CD54（ICAM-1）。相比之下，BJ成纤维细胞（CFSE阴性）是CD54阳性，但主要是负面的CD24。"https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52241/52241fig3highres.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 3.细胞表面染色<ol><li>标签管包括样品和关键控制（ 见表1）。 </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>管没有。 </td><td>样品名称</td><td>抗原荧光</td><td>稀释</td></tr><tr><td> 1 </td><td>未染色细胞</td><td> - </td><td></td></tr><tr><td> 2 </td><td>单染</td><td> CD24-APC </td><td> 1时50 </td></tr><tr><td> 3 </td><td>单染</td><td> TUJ1-的Alexa Fluor 488nm的</td><td> 1:2000 </td></tr><tr><td> 4 </td><td>双染</td><td> CD24-APC </td><td> 1:50 </td></tr><tr><td></td><td></td><td> TUJ1-的Alexa Fluor 488nm的</td><td> 1:2000 </td></tr><tr><td>五</td><td>单染</td><td>二级只:的Alexa Fluor 488nm的</td><td> 1:2000 </td></tr></tbody></table> 被包括在一个典型的流式细胞仪实验表1。L管IST。下表显示了一组所需的这个视频文章中描述的共染色实验样品管的最小的。一个理想的实验需要包括所有必要的控制（阴性，阳性以及补偿控制）对所获得的结果准确解释。 <ol start="2"><li>加入100微升细胞悬液（从2.7或1.3.2），以每次1.5 ml离心管。 注:确保最低0.1×10 6细胞是每100微升细胞悬液的存在。 </li><li>添加荧光团共轭抗体与样品在适当的稀释度。 注:确定工作稀释为在实验前各抗体。 见表2列表Ø˚F神经表面抗原。 </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>抗原</td><td>电池类型</td><td>参考</td></tr><tr><td> CD15 </td><td>神经干细胞</td><td> [28，67] </td></tr><tr><td> CD24 </td><td>神经细胞</td><td> [28，68] </td></tr><tr><td> CD29 </td><td>神经干细胞</td><td> [28，69，70] </td></tr><tr><td> CD44 </td><td>神经胶质细胞</td><td> [25] </td></tr><tr><td> CD49f的</td><td>神经干细胞</td><td> [38] </td></tr><tr><td> CD56（NCAM） </td><td>神经细胞</td><td> [71] </td></tr><tr><td> CD133 </td><td>神经干细胞</td><td> [27] </td></tr><tr><td> CD184 </td><td>神经干细胞和神经胶质细胞</td><td> [25] </td></tr><tr><td> CD200 </td><td>神经细胞</td><td> [38] </td></tr><tr><td> CD271 </td><td>神经嵴干细胞</td><tD&gt; [25] </td></tr><tr><td> A2B5 </td><td>神经胶质细胞</td><td> [31] </td></tr><tr><td> CORIN </td><td>多巴胺的前体</td><td> [35，36] </td></tr><tr><td> FORSE1 </td><td>神经干细胞（NS​​C） </td><td> [72] </td></tr><tr><td> GLAST </td><td>神经胶质细胞</td><td> [33] </td></tr><tr><td> NG2 </td><td>神经胶质细胞</td><td> [32] </td></tr></tbody></table> 表2.选择的神经表面抗原。此表提供发现由各种神经细胞类型被表示在举例用于表征神经谱系表面抗原的增加面板表面表位的列表。注意，这个选择是远未完成，大多数这些标记也通过一系列其他神经和非神经细胞中的表达。因此，将需要的几个标记物的组合，以更好地确定和分离指示神经子集。 <ol st艺术="4"&gt; <li>孵育在轨道摇床（200rpm）下在黑暗中30分钟。 </li><li>流缓冲区清洗<ol><li>加入1毫升的流缓冲到管中。离心机在380×g离心在4℃下4分钟。 </li><li>弃去上清液留下沉淀后面。 </li></ol></li><li>再次重复清洗步骤。 </li><li>第二次洗涤后，倒出上清液，并重新悬浮细胞在流缓冲器中以达到100μl的最终体积。 </li><li>使用样本流式细胞仪分析。另外，继续进行第4和第5。 注意:如果细胞是要排序并放回培养后的FACS（ 即没有固定或透化的活细胞悬浮液），收获，染色和分析步骤期间应用无菌技术。 </li></ol> 4.固定和透38 <ol><li>固定使用多聚甲醛（PFA）: <ol><li>制备含2％的PFA的PBS固定缓冲液中。 <br/&gt;注:PFA是对人体有害的环境。使用合适的个人防护装备和丢弃垃圾按照当地法规。 </li><li>加入500微升固定缓冲液100微升细胞悬浮液。 </li><li>孵育管在室温15分钟在定轨摇床（100rpm下）在黑暗中。 </li></ol></li><li> PBS洗: <ol><li>加入1毫升的PBS到管。离心机在380×g离心3分钟，在4℃。 </li><li>丢弃/倒出上清液留在管大约100微升。 </li></ol></li><li>透用Tween-20: <ol><li>制备含有0.7％吐温-20的PBS透化缓冲液中。 </li><li>加入500微升的透化缓冲液至100μl细胞悬浮液。 </li><li>孵育管在室温15分钟在定轨摇床（100rpm下）在黑暗中。 </li></ol></li><li>用PBS（如第4.2节 ），一旦洗细胞，并从协管去除上清mpletely，只留下沉淀的后面。 </li></ol> 5.细胞内抗原染色的38（图4） <ol><li>准备一抗的解决方案: <ol><li>稀释含1％牛血清白蛋白，10％血清（ 如正常驴或山羊血清）和0.5％吐温-20的PBS中稀释缓冲液中的一抗。 注:选择根据二次抗体中提出的物种中使用的血清。 </li><li>或者，根据制造商的说明使用第一抗体泽农荧光标记。 <ol><li>制备1微克的PBS初级抗体在适当的稀释液（总体积≤20微升）。 </li><li>添加5微升的Zenon IgG的荧光标记试剂（组分A）与抗体溶液。 </li><li>为5分钟，在RT下孵育所述混合物。 </li><li>添加5微升的Zenon封闭试剂（成分B）的反应混合物中。 </li><li>孵化为5分钟，在RT下将混合物。在30分钟内应用该抗体对所述样品。 </li></ol></li></ol></li><li>主要抗体染色: <ol><li>添加100微升的初级抗体溶液到细胞沉淀中，并轻轻磨碎混合。 </li><li>可替代地，泽农荧光素标记的抗体添加至细胞悬浮液在合适的稀释度。 </li><li>孵育管在室温30分钟在定轨摇床（200rpm）下，避光。用PBS洗一次细胞（如描述在第4.2节），并从完全管去除上清，只留下沉淀的后面。 </li></ol></li><li>第二抗体染色（不需要的泽农荧光素标记的抗体）: <ol><li>稀释在PBS中的第二抗体在适当的浓度。 </li><li>添加100微升的二次抗体溶液到细胞沉淀中，并轻轻磨碎混合。孵育管在RT在黑暗中30分钟在振荡器上（200rpm）下。 </li></ol></li><li>与PBS洗涤样品两次（如第4.2节 ）。 </li><li>与流缓冲洗一次（见第3.5节 ）。 </li><li>重新悬浮细胞在大约150微升流动缓冲液和流式细胞仪上的流动分析。 </li></ol><img alt="图4" src="/files/ftp_upload/52241/52241fig4highres.jpg" width="700" /> 图4.共染色的表面和细胞内的蛋白质。流式细胞术数据举例说明了主+二级抗体为基础的对与表面染色组合泽农荧光素为基础的细胞内染色的比较。在y轴（左上象限）和x轴（右下象限）独家阳性细胞显示染色MAP2和CD24，分别。后共染色，共用MAP2和CD24的表达可以看出，在右上象限（右图）。使用比较的Alexa Fluor 488（最高; AF488）与泽农荧光素（底）部MAP2标签产生类似的结果<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52241/52241fig4highres.jpg" target="_blank">，请点击这里查看该图的放大版本。</a> 6.流式细胞仪分析<ol><li>在完成使用流式细胞仪用适当的过滤器进行信号检测的染色方案后，立即进行流式细胞术分析。使用488nm的蓝色和640纳米的红色激光器FL-1（三十○分之五百三十三），FL-2（40分之585），和FL-4（25分之675）带通滤波器。 </li><li>建立基于前向和侧向散射不含碎片和死细胞的主要栅极。 </li><li>设置荧光门的表面和基于使用单一的彩色控制未染色样品，并赔偿频谱重叠细胞抗原≤0.5％。 </li></ol>

The authors declare no potential conflicts of interest.

这里介绍的协议是公认的用于从人干细胞来源的神经细胞培养物，但也可以同样适用于其他的神经细胞来源，包括原代组织或神经细胞系。除了 ​​胚胎来源，神经干细胞或祖细胞，可以提取自成人脑27的神经区域。此外，流式细胞仪和FACS可以被利用来量化，分 ​​析和分离各种细胞群体，包括成熟的神经元54，星形胶质细胞33，小胶质细胞55，少突56，或内皮细胞57从出生后的脑组织。 不管来源如何，对于实验每种细胞类型被确定为固定和透化需要的最佳条件，如过量的任一步骤的可能导致影响流式细胞读出散射特性的巨大变化。该协议的效率，还取决于STABIL表面分子的性张贴通透。应当注意的是，不同的表面抗原决定簇可以受细胞操作的程序，以不同程度的一部分。还酶促收获用胰蛋白酶更换或LIBERASE-1，例如，可能会影响到的范围内的细胞表面表位，但在较小的程度相比的Accutase或木瓜蛋白酶7的用法。 特定表位可能受相同的治疗范例在不同程度:虽然VCAM-1（血管细胞粘附蛋白-1; CD106）可受收获，解离，固定和透化在一个相当敏感的方式，ICAM-1（细胞间粘附分子1; CD54）可以显示在整个过程58保存一个更强大的表达模式。 而此前无论是表面25,28或细胞内的抗原标记14个神经生物学范例中得到应用，这两种方法结合米编制方法38可以产生新的神经元表面抗原候选人，并提供额外的流式细胞仪读数选项。 我们经常进行之前细胞内抗原染色表面染色的步骤，并已使用的策略，以扩大我们的一组表面标志物的使用范围的人类神经细胞系neuropoiesis 38。抗体和孵育时间的滴定将需要针对各个细胞类型进行优化，目标表位和实验范式。同样，CFSE浓度和每染色使用的细胞的数量必须为每个细胞系进行优化，因为不是所有的细胞系染色以相同的方式用CFSE浓度的相同的量。 CFSE具有494纳米的峰值激发和521纳米的峰值发射（绿色通道; FL-1检测器），因此，任何荧光溢出到其它信道需要被补偿，以确定特定的荧光发射。替代染料如铈LL跟踪紫（CTV），细胞增殖染料eFluor 670（CPD），地平线V550，V450地平线已成功尝试和测试，被用来代替CFSE 59,60。 在流式细胞仪中，以确定与背景的实际具体的荧光信号是至关重要的。因此，最适当的控制，必须仔细选择，并且可以包括使用非神经或其它阴性细胞系强调抗体特异性（所谓的内部阴性对照38,46）。每个单元的源的自体荧光，必须加以考虑。同型对照可以考虑主要是可有可无的，在大多数的神经细胞检测范式13,46。 定的相当大的变化在细胞培养系统中观察（特别是来自干细胞衍生的）和染色过程中，列入生物学重复的强烈推荐。应当指出的是遗​​传记者的用法是一个其他的方式来使用神经流式细胞61,62。最近，Maroof 等人使用一个Nkx2.1-GFP报道的方法，从人胚胎干细胞63鲜明类皮质神经元的亚群进行排序。 一个上崭露头角的领域是由生物医学领域等不同的肿瘤生物学和神经退行性疾病64神经外来体的探索代表和流外来体术或它们的隔离由珠为基础的分析提供了重要的工具，这个任务65。相关视频协议看66。

流式细胞仪已被广泛利用免疫学，血液学和肿瘤学，通过内在的散射特性，细胞表面的抗原表达，和其他荧光参数1-3定义细胞群。我们的见解血统发育和疾病有结果后，其最初的实现4,5个显著程度这一方法的不断细化。流的数量和整体分析潜在的意识提高术最近鼓励其在干细胞研究更广泛地使用，并且可以使在一个较短的时间框架6同样深刻的进展。然而，流式细胞术的应用，特别是分析和分离的神经种群早已被认为是具有挑战性的。在对比的是天然存在于悬浮液中的造血细胞，神经细胞类型通常由过度复杂的来源，其可包括神经胶质细胞和各种O收获疗法周围细胞以及过程承载神经元的一个复杂的网络。因此，神经生物学仍未实施流式细胞仪的多功能性，它在日常研究套路完整的潜力。然而，只要是可行的单细胞悬浮液可以产生（和协议已经设计和优化用于该目的7），流式细胞术和荧光激活细胞分选（FACS）可以被认为是在神经生物学分析剧目的一个有价值的元件8-11。 <img alt="图1" src="/files/ftp_upload/52241/52241fig1highres.jpg" width="600" /> 图1原理的流式细胞术分析和流量的部件仪的流式细胞仪包括三个主要系统:流体学，光学和电子学。细胞在悬浮液中简化的流被鞘flui完成（从主组织或离体培养的方法制备）通过水动力ð对焦，限制样品其中心的核心。该光学部件是由激光器照射细胞和光学过滤器，引导信号到相应的检测器的流。检测被转换成电子信号，然后由计算机处理和可视化的数据分析和门控在监视器上的光信号。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52241/52241fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 流式细胞方法利润的基本面，包括流式细胞仪的积木至少有一个基本的了解用户（审查见12,13;另参见图1）。的激光束相交一个流体动力学聚焦流体流包含该细胞悬浮液，这反过来又通过在"单一文件&#39;相继的激光束。该intercepti上的小区（或任何其它粒子，就此而言）与激光产生的光的，从该询问点的散射。散射光可在延续激光方向（前方散射，与颗粒的大小有关），垂直以及其方向来检测（侧向散射;反射颗粒/细胞的granulosity）。这些上述的散射特性不要求特定的标签，这是为什么未标记样品（或还细胞碎片，气泡等 ），将生成的二元前向散射与侧向散射图中常用的初始门控信号（事件）。通过使用适当的激光和特异于相应的激发和发射光谱过滤器，一个细胞可以为它的阳性，强度的水平，或不存在的荧光标记进行分析。大多数流式细胞应用已经通过细胞表面抗原的集中表征。不同于造血lineag即，在神经系一直根据表面抗原的表达模式5少广泛定义。利用表面抗原的一个优点是，活细胞能够进行细胞分选范式诸如FACS。与此相反，细胞内抗原染色，需要固定和透化步骤，以介导的表位 - 抗体相互作用，排除需要活菌下游应用。值得注意的是，这样的方法仍然允许众多的定量分析14以及下游分析为RNA和蛋白质的表达15。血液学，免疫学和肿瘤学经常用来十多标记结合来定义特定亚群16。此外，质谱术或CyTOF现在可以用来分析多达30个参数同时17,18。 对神经干细胞的应用，以及原代培养14,19,20细胞在异质体外是一种常见的现象21-23。不表示感兴趣的目标人群的细胞体现为实验读数24,25一个潜在的混杂因素。方便的是，在异构细胞悬浮液存在于不同的细胞亚群承受不同的（已知的或尚待解码）抗原的表达谱，这可以被用来确定这些不同的人群。流式细胞仪可因此在解决细胞异质性起到至关重要的作用，并且由此促进生物医学应用（ 体外测定法，细胞疗法），并通过集中最相关子集24,26上优化的定量读数。各种表面抗原组合已经确定，在过去几年中，以允许特定的神经细胞类型的定量和隔离。这包括CD133为神经干细胞的27中，CD15 / CD24 / CD29表面抗原的NSC的分离的组合，不同点的富集泰德神经元和神经嵴细胞28或CD15 / CD24 / CD44 / CD184 / CD271隔离神经胶质子集25，其他签名29,30之间。除了 ​​神经元，神经胶质标记包括A2B5 31，CD44 25，NG2 32和33 GLAST。最近的出版物开发的脑floorplate前体标记CORIN 34,35，以丰富的多巴胺的前体帕金森细胞移植范式36。光盘的分子不仅是标记物，但是细胞-细胞相互作用和当中的小区的能力功能相关介体从细胞外基质分子响应线索和生长因子37。进一步提高组合的CD抗原库表征神经系的发展策略之一是使用一种称为胞内标记筛选并确定CD抗原组合感兴趣的特定细胞类型。最近，我们利用这种方法，并确定了CD4部9f - / CD200 高组合的表达模式作为一种新的方法用于富集从neurally分化诱导多能干细胞培养系统38的神经元亚群。在这里，我们包括与讨论后者协议（和可选的变体），其中表面染色和细胞内染色，可同时用于通过流式细胞术确定的神经细胞亚群。 <img alt="图2" src="/files/ftp_upload/52241/52241fig2highres.jpg" width="300" /> 实验协议选项图2的流程图。该图描述中涉及的协议的主要步骤的示意性表示。可选步骤（CFSE染料或细胞内的抗原标记）被浅灰色方框表示。收获后，有必要进行评估之前的细胞表面染色的神经细胞悬浮液的活力和细胞数目。积极作为以及阴性对照需要包含除所关注的样品。样品可以通过流式细胞仪进行分析和/或细胞分选范例使用。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52241/52241fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 虽然我们以前使用与二次抗体结合第一抗体细胞内染色38，我们现在通过荧光Fab片段（泽农标签），为轻微的变化引进第一抗体的非共价标记，从而减少了细胞操作39的步骤。此外，作为协议的通用性的又一个例子中，我们通过羧基琥珀酰亚胺酯（CFSE）表面抗原染色前使用的一个实验性的子集的可选标记。这样的CFSE预标记使两个细胞系或实验条件下立即直接比较（CFSE标记的VS。未标记的）单一样品管，减少变异或孵化时间的细微差异，节约抗体内。 CFSE是通常用于细胞跟踪40，在增殖41,42和条形编码实验43,44既定荧光染料。最后，虽然实际的分拣步骤（FACS，免疫细胞分离或immunopanning）不是该协议的一部分，在原则上，此处描述的收集和标识程序做，可以进行表面抗原或细胞内标记为基础分类应用收率样品15 25,28。 在本文中，我们的目标是:总结出可行的表面抗原染色协议25,28，总结了检测细胞内目标的协议，以及结合表面和细胞内的抗原分析38，出现细胞内CFSE染料标记步骤41,45作为实验选项COMparative分析神经细胞群，总结方法流式细胞仪分析（适当控制13,46，门控策略和数据呈现47）。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:456px;">DMEM/F12 (1:1) (Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12)</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:124px;">11330057</td>
			<td style="width:149px;">www.lifetechnologies.com</td>
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		<tr height="19">
			<td height="19" style="height:19px;">DPBS without Ca2+ Mg2+</td>
			<td>Life Technologies</td>
			<td>14190169</td>
			<td>www.lifetechnologies.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal bovine serum, qualified, E.U.-approved, South America origin (FBS)</td>
			<td>Life Technologies</td>
			<td>10270-106</td>
			<td>www.lifetechnologies.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MEM Non-essential amino acids (100 x)</td>
			<td>Life Technologies</td>
			<td>11140035</td>
			<td>www.lifetechnologies.com</td>
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		<tr height="17">
			<td height="17" style="height:17px;">TrypLE Express</td>
			<td>Life Technologies</td>
			<td>12604013</td>
			<td>www.lifetechnologies.com</td>
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		<tr height="17">
			<td height="17" style="height:17px;">Trypan blue solution, 0.4%</td>
			<td>Life Technologies</td>
			<td>15250061</td>
			<td>www.lifetechnologies.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Paraformaldehyde</td>
			<td>Carl Roth</td>
			<td>335.3</td>
			<td>www.carlroth.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bovine serum albumin (BSA) Fraction V</td>
			<td>PAA Laboratories, Coelbe</td>
			<td>K41-001</td>
			<td>www.gelifesciences.com</td>
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		<tr height="17">
			<td height="17" style="height:17px;">Tween-20 Detergent</td>
			<td>Calbiochem</td>
			<td>655205</td>
			<td>www.merckmillipore.de</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Carboxyfluorescein succinimidyl ester (CFSE)</td>
			<td>eBioscience</td>
			<td>65-0850-84</td>
			<td>www.ebioscience.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DMSO</td>
			<td>AppliChem</td>
			<td>A1584</td>
			<td>www.applichem.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bottle top filters express plus 0.22 &#181;m, 250 ml</td>
			<td>Millipore</td>
			<td>SCGPU02RE</td>
			<td>www.millipore.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cell culture treated flasks (T 25)</td>
			<td>NUNC</td>
			<td>156367</td>
			<td>www.thermoscientific.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cell culture treated flasks (T 75)</td>
			<td>NUNC</td>
			<td>156499</td>
			<td>www.thermoscientific.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Conical tubes (15 ml)</td>
			<td>Greiner Bio-One</td>
			<td>188271</td>
			<td>www.greinerbioone.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Conical tubes (50 ml)</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
			<td>www.greinerbioone.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pasteur pipet, glass (150 mm)</td>
			<td>STEIN Labortechnik, Remchingen</td>
			<td>S03710150</td>
			<td>www.stein-labortechnik.de</td>
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		<tr height="17">
			<td height="17" style="height:17px;">Pipet tips (0.1-10 &#181;l)</td>
			<td>Corning</td>
			<td>4125</td>
			<td>www.corning.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pipet tips (1-200 &#181;l)</td>
			<td>Corning</td>
			<td>4126</td>
			<td>www.corning.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pipet tips (100-1000 &#181;l)</td>
			<td>Corning</td>
			<td>4129</td>
			<td>www.corning.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Serological pipets, 5 ml</td>
			<td>Corning</td>
			<td>4051</td>
			<td>www.corning.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Serological pipets, 10 ml</td>
			<td>Corning</td>
			<td>4101</td>
			<td>www.corning.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Serological pipets, 25 ml</td>
			<td>Corning</td>
			<td>4251</td>
			<td>www.corning.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Microcentrifuge tubes (0.5 ml)</td>
			<td>Sarstedt</td>
			<td>72,699</td>
			<td>www.sarstedt.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Microcentrifuge tubes (1.5 ml)</td>
			<td>Greiner Bio-One</td>
			<td>616201</td>
			<td>www.greinerbioone.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Microcentrifuge tubes (2.0 ml)</td>
			<td>Sarstedt</td>
			<td>72,695,500</td>
			<td>www.sarstedt.com</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Human CD24 APC monoclonal antibody</td>
			<td>eBioscience</td>
			<td>17-0247-42</td>
			<td>www.ebioscience.com 
			Working dilution 1:50</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Human CD54 PE monoclonal antibody</td>
			<td>eBioscience</td>
			<td>12-0549-42</td>
			<td>www.ebioscience.com 
			Working dilution 1:50</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:456px;">Neuronal Class III &#946;-Tubulin (Tuj1) polyclonal antibody</td>
			<td>Covance</td>
			<td>PRB-435P</td>
			<td>www.covance.com 
			Working dilution 1:2000</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Alexa Fluor 488 Donkey anti Rabbit</td>
			<td>Life Technologies</td>
			<td>A21206</td>
			<td>www.lifetechnologies.com 
			Working dilution 1:2000</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Zenon&#174; Fluorescein Rabbit IgG Labeling Kit</td>
			<td>Life Technologies</td>
			<td>Z-25342</td>
			<td>www.lifetechnologies.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Neubauer-Improved counting chamber</td>
			<td>Marienfeld</td>
			<td>0640010</td>
			<td>www.marienfeld-superior.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vortex</td>
			<td>Scientific Industries</td>
			<td>G560E</td>
			<td>www.scientificindustries.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Thermomixer comfort</td>
			<td>Eppendorf</td>
			<td>5355 000.001</td>
			<td>www.eppendorf.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Accuri C6 flow cytometer</td>
			<td>Becton Dickinson (BD)</td>
			<td align="right">653118</td>
			<td>www.bdbiosciences.com/instruments/accuri</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Microcentrifuge refrigerated, PerfectSpin 24 R</td>
			<td>Peqlab</td>
			<td>91-PSPIN-24R</td>
			<td>www.peqlab.de</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Orbital shaker, Unimax 1010</td>
			<td>Heidolph</td>
			<td>543-12310-00</td>
			<td>www.heidolph-instruments.de</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Centrifuge refrigerated, Rotanta 96 RC</td>
			<td>Hettich</td>
			<td>4480-50</td>
			<td>www.hettichlab.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Class II Biological safety cabinet Safe 2020</td>
			<td>Thermo Scientific</td>
			<td>51026640</td>
			<td>www.thermoscientific.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CO2 Incubator, Heracell 240i</td>
			<td>Thermo Scientific</td>
			<td align="right">51026331</td>
			<td>www.thermoscientific.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vacuum system, Vacusafe comfort</td>
			<td>Integra Biosciences</td>
			<td align="right">158320</td>
			<td>www.integra-biosciences.de</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Microscope, Axiovert 40 CFL</td>
			<td>Zeiss</td>
			<td align="right">451212</td>
			<td>www.zeiss.de</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pipet controller, accu-jet pro</td>
			<td>Brand</td>
			<td align="right">26303</td>
			<td>www.brand.de</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Micropipet, Pipetman neo P20N (2-20 &#181;l)</td>
			<td>Gilson</td>
			<td>F144563</td>
			<td>www.gilson.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Micropipet, Pipetman neo P200N (20-200 &#181;l)</td>
			<td>Gilson</td>
			<td>F144565</td>
			<td>www.gilson.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Micropipet, Pipetman neo P1000N (100-1000 &#181;l)</td>
			<td>Gilson</td>
			<td>F144566</td>
			<td>www.gilson.com</td>
		</tr>
	</tbody>
</table>

flow cytometry protocols for surface and intracellular antigen analyses of neural cell types

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology.

这里介绍的协议允许多功能的实验方法（ 图2）。在其最短的版本（ 步骤1，3和6），它可以被认为是表面抗原的简单染色的指南。在它的更复杂的形式，一些共标记的范例与一系列胞内抗原可以追求（任选的步骤2和/或4至5）。此外，将CFSE标记步骤举例说明它的应用程序，用于细胞-细胞相互作用的研究细胞标记/亚群的跟踪或流式细胞条码实验（ 步骤2）。存活细胞群体（来自步骤2获得和/或3）是适合于细胞分拣范式（FACS）和下游分析，包括后排序细胞培养物。另一方面，将合并的细胞内和细胞表面抗原染色（ 步骤3至5）提供在自己的权利的分析读出的机会。例如，确定共定位于神经细胞群的特定子集某些CD抗原（由我们Turaç 等 38应用到识别CD49f的- 。/ CD200 高签名神经FACS）。这些和其他实施例的神经标记物在表2中 ，其中的CD15，CD29，CD49f的和FORSE1，主要与神经干细胞相关联提供，并CD24和CD56是丰富对神经元细胞。虽然少数这些标记是排他性的，不同的染色强度和多元表达分析可以帮助确定具体的指标集分析和特定的神经细胞亚群的隔离。 图3例示了从细胞内的染料和表面染色的组合使用获得的典型结果。这些数据显示了如何两个不同的细胞群体可容易地区别我呐混合的样品，由于人口的1的现有标记。这里，SH-SY5Y成神经细胞瘤细胞用CFSE标记，并在绿色通道（FL1; y轴）显示出荧光，这显然是由未染色的第二群体中分离出来，在这种情况下，BJ成纤维细胞。而完善的特性的成纤维细胞（CD54）和神经（CD24）标记显示（存在于它们各自的目标人群; FL2或FL4荧光，分别; x轴）的类似方法可以被利用来筛选额外不明抗原。此外，那被标记之前共培养实验的两个（或更多个）的亚群的细胞 - 细胞相互作用的影响，可以研究以这种方式。 图4显示了未染色的SH-SY5Y细胞，以及固定和透化后染色CD24-APC和/或细胞内的神经元MAP2抗原（FITC通道）样本。因为后者会影响散射特性，巴ckground荧光和表面抗原染色，该地块成为细胞内染色后记录神经CD24的保持表面表达。此外，示出了共定位对CD24阳性级分的神经元MAP2染色。以确保真实的检测表面抗原通过比较CD24表面表达的活细胞是至关重要的。的细胞内检测超过背景此外特异性的，非特异性抗体结合和自体荧光有待证实。检测经由泽农基于荧光的或初级+二级抗体为基础的策略的细胞内抗原的产生可比较的结果，这提供了考虑这多个时有效的方法（跳过步骤5.3）的基本原理。而该协议是健壮并允许一系列表面和胞内标志物具有有限的背景荧光的联合检测，建议密切附着在孵育时间和清洗步骤在协议中提供的。此外，最好是分析的至少三个独立实验的重复，并注意，标记强度和表面表位的稳定性可以是标志物特异性，并且可能需要进一步优化。一旦建立了一个特定的目标，尽管缺乏分析形态特征的能力，流式细胞仪检测表面和胞内抗原或细胞内抗原单独是与常规荧光显微镜兼容。

Watch this Scientific Journal Video about Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types at JoVE.com

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.

神经细胞类型的流式细胞仪协议的表面和细胞内抗原分析

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of ...

flow-cytometry-protocols-for-surface-intracellular-antigen-analyses

Nanyang Technological University

Research

JoVE Journal

Neuroscience

52.0K 次观看.  University of Freiburg. We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.

视频: Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

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 ...

科研

神经科学

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 ...

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 ...

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.

Our laboratory developed a novel high-throughput automated imaging system that is useful for the detection of several different behaviors in 7-day-old zebrafish larvae. The system can be used for detecting subtle changes in behavior after the larvae have been exposed to environmental toxicants or pharmaceuticals.

在斑马鱼幼虫的高通量自动化的行为分析

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system ...

Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

Traditional methods of immunohistochemistry (IHC) following tissue fixation allow visualization of various cell types. These typically proceed with the application of antibodies to bind antigens and identify cells with characteristics that are a function of the inherent biology and development. Adult hippocampal neurogenesis is a sequential process wherein a quiescent neural stem cell can become activated and proceed through stages of proliferation, differentiation, maturation and functional integration. Each phase is distinct with a characteristic morphology and upregulation of genes. Identification of these phases is important to understand the regulatory mechanisms at play and any alterations in this process that underlie the pathophysiology of debilitating disorders. Our heat-induced antigen retrieval approach improves the intensity of the signal that is detected and allows correct identification of the progenitor cell type. As discussed in this paper, it especially allows us to circumvent current problems in detection of certain progenitor cell types.

During adult hippocampal neurogenesis, a distinct set of genetic markers are expressed when a quiescent neural stem cell sequentially progresses and develops into a functionally integrated neuron in the circuit. Using heat-induced antigen retrieval, progenitor cell types that are otherwise difficult to detect are identified with improved effectiveness.

Traditional  methods of immunohistochemistry (IHC) following tissue fixation allow visualization  of various cell types. These typically proceed with the ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

用优化的n-甲基 d-葡乙胺保护恢复法制备急性脑切片

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

使用克隆测定神经干细胞的计数。

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

人视网膜色素上皮细胞向大鼠眼巩膜视网膜移植精制方案的研制

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

时间依赖性增加对微电极阵列刺激神经元细胞培养物的网络响应

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.