这项工作是从斯特里特基金会的资金支持。

1。细胞培养之前基本设置<ol><li>国科会NeuroCult基础培养基和NeuroCult国科会扩散补充，以9:1的比例混合，分别作出完整的国科会介质。 </li><li> 37℃水浴用于热身介质。 </li><li>适量热灭活胎牛血清（FCS）的解冻。 </li><li>适量股票生长因子解决方案，包括表皮生长因子（表皮生长因子在10微克/毫升），碱性成纤维细胞生长因子（b-FGF的10微克/毫升）和0.2％肝素溶液磷酸缓冲液（PBS）解冻。 </li><li>组织培养瓶（的T25，T75和T175）国科会扩大和分化的需要。 </li><li>组织培养处理96和24孔板和盖玻片电镀排序细胞所需要的。 </li></ol> 2。隔离和扩展的神经干细胞（传代） <ol><li>神经干细胞和祖细胞的分离和扩大小鼠B下雨作为单独的协议1 2 3描述。 </li><li>当神经球（小学或传代神经球）是亚文化（直径在150-200微米），培养基的领域转移到一个适当大小的无菌组织培养管，离心5分钟，在800转（110克）在室温下。 </li><li>培养上清液中被删除，球都在重新悬浮预热的0.05％胰蛋白酶EDTA 1毫升。 </li><li>培养在37℃水浴2-3分钟后，大豆胰蛋白酶抑制剂的加入同等体积的细胞悬液，以阻止胰蛋白酶的活性。 </li><li>为了确保已完全灭活的胰蛋白酶，轻轻洗涤后的细胞悬液，下降了3-5倍。 </li><li>在800转（110克），5分钟后离心，上清液被删除，在1毫升完整的国科会培养基重新悬浮细胞。 </li><li> 90μl的trypa的混合细胞悬液10μLñ蓝进行细胞计数。 注：也可用于其他合适的细胞稀释。 </li><li>继代1，2或分化的细胞，然后使用如下所述。 </li></ol> 3。神经干细胞和祖细胞的分化， 神经母细胞含量（NBA的） <ol><li>分离神经球的细胞被镀在2-3×10 5细胞/毫升完整的国科会20毫微克/毫升表皮生长因子，10 ng / ml的碱性成纤维细胞生长因子，1微升/毫升0.2％肝素和5％的培养基中的浓度。热灭活在一个适当大小的组织培养瓶中FCS。 5毫升用于介质的T25，20毫升的T75和T175瓶40毫升。 注 ：FCS将导致细胞牢固地连接到基板和有没有需要对NBA文化的涂层烧瓶。 </li><li>邪教余吕杏茜瓶，然后在37℃培养箱孵化培养，用5％的CO 2为3-4天。这一时期被称为增殖阶段 ，在此期间，国科会的后代基板的重视，为单层细胞增殖。 </li><li>当文化成为90-95％汇合，每个烧瓶介质切换到完整的国科会与5％胎牛血清培养基生长因子没有任何补充。 </li><li>再次，培养瓶在37℃培养箱孵化培养，用5％的CO 2，另一3-4天。这一时期被称为分化阶段 ，其间的神经祖细胞星形细胞单层上出现和增殖，使未成熟的神经细胞的殖民地。分化阶段4-5天，文化是分离的神经母细胞采用流式细胞仪技术准备。 </li></ol> 4。流式细胞仪对细胞的制备 trypsinization <ol><li>区分国科会的后代分化阶段4-5天洗一次文化与适量的PBS（2毫升，4毫升的T75的T25和T175瓶8毫升）从文化FCS。 注：FCS的块胰蛋白酶-EDTA的活动。 </li><li>然后，足量预温的0.05％胰蛋白酶-EDTA（2毫升，4毫升的T75的T25和T175瓶8毫升）添加到每个烧瓶覆盖单层细胞培养在1-2分钟37°C间湿润的孵化器。 </li><li>烧瓶中被选中，在显微镜下评估胰蛋白酶-EDTA对文化的影响。烧瓶中，然后用一只手，并举行袭击另一方面坚决驱逐从烧瓶底部的细胞。 </li><li>添加到等量的大豆胰蛋白酶抑制剂瓶解渴胰蛋白酶的活性的。细胞悬液轻轻吸管上下，以确保胰蛋白酶失活，并实现了均匀的单细胞停牌。 注：另外，5％胎牛血清培养基可以用来解渴胰蛋白酶的活性。 </li><li>要删除非游离团块，细胞悬液通过40微米大小的滤网。 </li><li>细胞悬液，然后在5分钟800转（110克）离心，上清液被删除，并在适当的音量一个完整的国科会中小型重新悬浮细胞。 </li><li>调整的最终体积细胞悬液，使细胞密度不超过2-3X10 6 -cells/ml。高密度可能会导致在流式细胞仪机流堵塞。 </li><li>如果您想隔离神经细胞，根据其物理性质，化学性质，只是，现在你可以进行排序为第4部分中描述的流式细胞仪。 </li><li>在排序之前，碘化丙啶（PI，Sigma公司，500微克/毫升的PBS）1微升/毫升细胞悬液，以排除死细胞的浓度增加。 </li></ol>住CEL升免疫标记要达到的纯度较高的神经细胞，收获单细胞可以从NBA文化的PSA-NCAM的（早期不成熟的神经祖细胞的标志），然后从神经元的人口阳性细胞染色可以使用流式细胞仪机分离。 <ol><li>从NBA文化的单细胞悬液进行细胞计数后，分成四组： <ul><li>细胞组，作为调整控制参数和门（0.5-1×10 6细胞）。 </li><li>细胞加PI，作为调整控制参数和门（0.5-10×10 6细胞）。从本组PI阴性细胞也可以排序基于物理特性的细胞。 </li><li>中学单（同型对照组），也为调整控制参数和门（1-2×10 6细胞）。 </li><li>免疫标记组（8-10×10 6个细胞），是为PSA-NCAM的染色来实现未成熟的神经细胞的进一步净化。排除经分拣，死细胞，PI以及添加到该组。 </li></ul></li><li>要启动免疫标记，细胞离心5分钟800转（110克）。除去上清100μL完整的国科会中小型重新悬浮细胞。 </li><li>藻红蛋白（PE）标记的抗PSA-NCAM的抗体添加比例的10微升/ 5-10×10 6细胞被染色的PSA-NCAM的细胞悬液。以同样的方式，被添加到一个PE标记的小鼠IgG1的同型对照抗体的同型对照细胞悬液。按照指示要使用的抗体浓度抗体规范表。细胞悬浮液，然后轻轻混匀，室温孵育10-15分钟，在黑暗。 </li><li>细胞悬液（PSA-NCAM和同型对照管），然后在5分钟800转（110克），离心，上清液被删除，细胞重新-suspended在1毫升完整的国科会介质。 </li><li>从样本中删除多余的联合国有限的抗体，第4步重复2-3次。 </li><li>经过重新悬浮细胞，在适当的介质体积碘化丙啶（PI，Sigma公司，在PBS的500微克/毫升），加入1微升/毫升细胞悬液（细胞加PI，同型控制和浓度的PSA NCAM的染色样品），以排除死细胞，流式细胞仪分析。 </li><li> 15毫升无菌猎鹰管完全培养基中含有1毫升准备收集排序从不同的细胞群的细胞。 </li></ol> 5。荧光激活细胞分类（FACS）对未成熟神经元<ol><li>流式细胞仪机设置基于其用户手册通过流式细胞仪设备专业技术人员的指令。 </li><li>磷酸盐缓冲液（PBS）或任何其他适当的解决方案dependin制造商的指示克，可以作为鞘液在28 PSI通过90微米的喷嘴。 </li><li>调整系统上的压力差，这样的排序触发率不超过2500次/秒。 </li><li>从单细胞的细胞悬液组运行通过流式细胞机。 </li><li>首先，细胞（事件）策划，根据他们的大小（前向散射光（FSC）的）与内部的复杂性（侧散射（SSC）光）来区分不同的细胞群。这样做，FSC和SSC电压参数应进行调整，使细胞可以在流式细胞仪的阴谋。 </li><li>为了排除细胞团块和双峰，细胞首先绘制基于前向散射面积（FSC - A）与前向散射脉冲宽度（FSC瓦）和单细胞人口人口1（P1）的门。然后绘制的P1细胞的基础上侧散射（SSC - A）区与侧散射脉冲宽度（FSC瓦），这个时间的单细胞GAT人口2（P2）。在建立连续这两个闸门，团块或双峰（高FSC脉冲宽度和高SSC脉冲宽度）被排除在外。 </li><li>排除死亡或受损的细胞，单细胞（P2）的绘制基于PI与FSC标准和单个活细胞的数量是门的反应。 </li><li>基于FSC与SSC的绘制单个活细胞来区分不同的细胞群体，根据细胞的大小和内部粒度。在这个阶段，两个主要的细胞群的门FSC 低南南合作低 （P3）和FSC 高南南合作高 （P4）的细胞群。 </li><li> PSA-NCAM免疫反应未成熟的神经细胞来自FSC的低 SSC 低人口，首先是FSC的低 SSC从控制人口低 （加PI和同型对照细胞）排序绘制基于体育阳性的与金管会的一个负细胞门（小五），然后从PSA-NCAM的阳性细胞染色组是作为人口6（六）门。 </li><li>细胞的利益调整所有需要的大门后，整理成15毫升无菌组织培养含1毫升国科会介质管。 </li><li>排序的细胞，然后离心5分钟1200转（240克）。 </li><li>被丢弃上清和沉淀重新悬浮在适当体积中等（颗粒大小而定）和细胞计数执行。 </li><li>细胞，然后镀在聚-L-鸟氨酸涂层的96孔板/ 200-250μl完整的国科会介质的密度在20-30×10 3细胞，用5％FCS和20 ng / ml的重组人BMP4的5 。 </li></ol> 注：大衣96孔，100μL聚-L-鸟氨酸的工作解决方案（1.5毫升聚-O和8.5毫升无菌PBS），每孔加入板至少在37℃培养箱培养小时。然后，聚邻删除，每口井洗3次，用无菌PBS（让我们留在PBS以及在每个时间为10分钟）。使用聚-L-鸟氨酸的工作方案/如果使用24孔板500μL。 <ol start="14"><li>固定细胞，然后在不同时间点后排序，用冷的4％多聚甲醛和适当的神经元和神经胶质细胞标志物免疫组化染色评估排序从不同的细胞群的细胞表型。 </li></ol> 6。代表结果在神经母细胞检测（NBA）的分化培养，重视组织文化基板镀细胞成长为单层。根据初始细胞接种密度，文化将打开到90-95％汇合文化后3-4天。在这个阶段，主要是星形细胞单层可以是与小圆4,5顶部的神经祖细胞（ 图1）可视化下。切换中一种生长因子的培养基后，神经祖细胞开始除以迅速，并在4-5天的星形细胞单层（ 图2）上的未成熟神经母细胞产生的集群。流游离NBA文化的流式细胞仪分析分化阶段4-5天，显示了两个主要的细胞群; FSC 低南南合作低和FSC 高南南合作高根据细胞大小（FSC）和内部粒度（SSC）（ 图3）。排序细胞的免疫荧光分析表明，在FSC的低 SSC（超过75％，其中表达β-Ⅲ微管蛋白） 低人口主要位于神经细胞，而细胞排序从FSC 高 SSC 高人口大多阳性星形胶质细胞GFAP（ 图4）。未成熟的神经细胞的进一步净化了附近同质性（97％）可以实现的PSA-NCAM的红外细胞正选择从FSC 低 SSC 低人口（ 图3，4）。从E14小鼠神经节隆起分离出神经干细胞生成的丰富的神经细胞获得1 GABA能表型分化和后快递的DARPP-32，中棘神经元的标志，（ 图5）。 <img src="/files/ftp_upload/3712/3712fig1.jpg" alt="图1"/> 图1。神经母细胞从E14小鼠神经干细胞增殖期4天的检测文化。此单层培养由平坦的星形细胞（箭头）下一轮的神经祖细胞的顶端（箭头）。原放大倍率20倍。 <img src="/files/ftp_upload/3712/3712fig2.jpg" alt="图2"/> ）相衬，B）的免疫荧光染色： 图2神经母细胞从E14小鼠神经干细胞分化阶段4天的检测文化。 在 A注意未成熟的神经细胞（集群箭头</stro吴和βIII 在 B微管蛋白染色星形细胞单层的顶端（箭头，GFAP在B染色））。原放大倍率20倍。 <img src="/files/ftp_upload/3712/3712fig3.jpg" alt="图3"/> 图3代表排序图：A） FSC与SSC的排序前的双峰和死细胞的排斥情节，B，C）。排序地块双峰排除基于FSC和SSC脉冲宽度，D）。基于碘化丙啶免疫排斥死亡，受损细胞E）。 FSC与SSC的双峰和死亡的细胞排斥的阴谋后，显示标记作为FSC 低 SSC 低 ，FSC 高南南合作高人口的两个主要群体，F，G）的 ​​。排序地块隔离从FSC 低 SSC 低人口的PSA-NCAM的红外未成熟细胞。 图4。代表从细胞显微照片从不同的人群排序电镀后的一天。）FSC的低 SSC的低人口（超过75％，这些细胞是神经元）。 二 ）FSC的高 SSC的高人口（大多数这些细胞是星形胶质细胞）C）的PSA-NCAM的排序从FSC 低 SSC的低人口（几乎所有的排序细胞是神经元）的细胞。原来放大20倍。 <img src="/files/ftp_upload/3712/3712fig5.jpg" alt="图5"/> 图5。排序未成熟的神经细胞分化为GABA能神经元（A）和明示的DARPP-32（B）中等棘神经元的标志。

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作者没有透露。

隔离定义的神经细胞群（即神经元，星形胶质细胞和少突胶质细胞）的能力可能会解决一些与临床应用神经干细胞（NS​​Cs）的相关障碍。首先，它使我们的供体细胞的人口开始充分体现。第二，它可以让我们使用一个特定的细胞类型或不同类型的细胞组合，与特定的比例，根据疾病的性质或阶段。最后，使用高浓缩铀的预分化的神经细胞的祖细胞，可以显着降低可能不受控制地增殖和肿瘤的形...

<table border="1"><tr><td> 试剂名称 </td><td> 类型 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> NeuroCult国科会的基础培养基</td><td>中等</td><td>干细胞技术</td><td> 05700 </td><td></td></tr><tr><td> NeuroCult国科会扩散补充</td><td>中等补充</td><td>干细胞技术</td><td> 05701 </td><td></td></tr><tr><td> ％0.05胰蛋白酶EDTA </td><td>试剂</td><td> Gibco公司</td><td> 25300-062 </td><td></td></tr><tr><td>大豆胰蛋白酶抑制剂</td><td>试剂</td><td>西格玛</td><td> T6522 </td><td></td></tr><tr><td>笔/链球菌</td><td>试剂</td><td> Gibco公司</td><td> 15140-122 </td><td></td></tr><tr><td> *的MEM </td><td>试剂</td><td> Gibco公司</td><td> 41500-018 </td><td> HEM的组成部分</td></tr><tr><td> *肝素钠</td><td>试剂</td><td>西格玛</td><td> H4034 </td><td> HEM的组成部分</td></tr><tr><td> *蒸馏水</td><td>试剂</td><td> Gibco公司</td><td> 15230-147 </td><td></td></tr><tr><td>细胞过滤器</td><td>筛</td><td>屋宇署猎鹰</td><td> 352340 </td><td></td></tr><tr><td>的T25瓶</td><td>文化洁具</td><td> nalge NUNC国际</td><td> 136196 </td><td></td></tr><tr><td> T80瓶</td><td>文化洁具</td><td> nalge NUNC国际</td><td> 178905 </td><td></td></tr><tr><td> 15毫升管</td><td>文化洁具</td><td>屋宇署猎鹰</td><td> 352096 </td><td></td></tr><tr><td> 50毫升管</td><td>文化洁具</td><td>屋宇署猎鹰</td><td> 352070 </td><td&gt; </td></tr><tr><td>表皮生长因子</td><td>生长因子</td><td>研发</td><td> 2028-EG </td><td></td></tr><tr><td> B-FGF </td><td>生长因子</td><td>研发</td><td> 3139-FB： </td><td></td></tr><tr><td>肝素</td><td>生长因子</td><td>西格玛</td><td> H4784 </td><td>重组在PBS </td></tr><tr><td>抗PSA-NCAM的-PE </td><td>抗体</td><td>美天旎生物技术</td><td> 130-093-274 </td><td></td></tr><tr><td> PE标记的鼠IgG1 </td><td>同型对照抗体</td><td>屋宇署Pharminogen </td><td> 556029 </td><td></td></tr><tr><td>肝素</td><td>生长因子</td><td>西格玛</td><td> H4784 </td><td>重组在PBS </td></tr><tr><td> HrBMP-4 </td><td>生长因子</td><td>研发</td><td> 314-BP-010 </td><td></td></tr><tr><td>聚-L-Ornit海因</td><td>试剂</td><td>西格玛</td><td> P4957 </td><td></td></tr><tr><td>胎牛血清</td><td>中等补编</td><td> Gibco公司</td><td> 10099-141 </td><td></td></tr><tr><td> GFAP的</td><td>抗体</td><td> DAKO公司</td><td> Z0334 </td><td></td></tr><tr><td> B - 三微管蛋白</td><td>抗体</td><td> Promega公司</td><td> G7121 </td><td></td></tr></table> *为了使下摆，搭配1的MEM的X 10升包，1M肝素钠和160毫升，并带来8.75 L使用蒸馏水的体积。设置最终的pH值至7.4，并储存在4°C

the neuroblast assay: an assay for the generation and enrichment of neuronal progenitor cells from differentiating neural stem cell progeny using flow cytometry

神经干细胞（NS​​Cs）可以分离和扩大规模大，使用的神经球法和中枢神经系统（CNS）的三个主要类型的细胞分化，即星形胶质细胞，少突胶质细胞和神经元。这些特点使神经干细胞和祖细胞在体外研究，如药物筛选，神经毒理学和电，也为许多神经系统疾病的细胞替代疗法的一个宝贵的可再生的细胞来源。然而，在实践中，国科会的后代，低生产的神经元和少突胶质细胞和星形胶质细胞的分化限制其临床应用后优势的异质性。在这里，我们描述了一个新颖的方法，利用荧光激活细胞分选（FACS）技术，从国科会的后代小鼠未成熟神经元的生成和随后净化。使用这种方法，一个非常丰富的神经祖细胞的人口可达到机智豪特任何明显的星形胶质细胞和真正的国科会污染。该程序包括分化E14小鼠神经节隆起，使用的神经球检测其物理（大小和内部的复杂性）和荧光性能，使用流式细胞仪技术的基础上，由未成熟的神经细胞的分离和富集分离和扩大NSC的后代。总体而言，它需要5-7天，产生神经球和6-8天来区分国科会的后代和分离高纯度未成熟的神经细胞。

Watch this Scientific Journal Video about The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytomet

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

这个视频协议，表明了新方法的产生和后续的纯化，根据自己的身体（大小和内部的粒度）和荧光性能，使用流式细胞仪技术从可再生来源的神经干细胞（NS​​Cs）的神经祖细胞。

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 ...

the-neuroblast-assay-an-assay-for-generation-enrichment-neuronal

Research

JoVE Journal

Neuroscience

15.4K 次观看.  The University of Florida. 这个视频协议，表明了新方法的产生和后续的纯化，根据自己的身体（大小和内部的粒度）和荧光性能，使用流式细胞仪技术从可再生来源的神经干细胞（NS​​Cs）的神经祖细胞。

视频: The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

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). ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

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 ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

从人多能神经嵴前体细胞的无饲养推导干细胞

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

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.

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 ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

神经干细胞和祖细胞来自成年小鼠脑室下区细胞分选和实时成像的细胞周期动力学

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

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. ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

通过Sialoglycan的代谢标记识别主要神经干细胞和祖细胞的细胞表面标记

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...