这项工作的经费由自然科学和工程研究理事会，加拿大（拨款＃249669，＃482986）和加拿大心脏及中风基金会（拨款＃485508）。作者感谢优素福埃尔 - 哈耶克和博士齐哇嗯为他们的协助开发实验的协议。

多伦多大学的动物保健委员会批准了在所有涉及动物处理的程序，按照制度指引（协议。20009387）。应当执行下面的方法，使用无菌的工具和技术，在层流罩（如适用）。 在下面的协议文本中，短语“EFH-SFM”是指以与表皮生长因子，碱性成纤维细胞生长因子和肝素的无血清培养基中。 EFH-SFM调查时，未分化的NPC，因为这些有丝分裂原的趋电维持其未分化状态的NPC 4。当调查趋电的NPC诱导分化成成熟的细胞类型，“FBS-SFM”指的是含1％胎牛血清的无血清培养基中。 FBS促进NPC的分化为成熟的神经表型。 1。神经前体细胞的分离和培养（不视频中所示） <ol><li>一个CD1小鼠（6-8周龄）麻醉与异氟烷和牺牲精神，通过颈椎脱位。 </li><li>浇在70％的乙醇和锐性剥离剪刀斩首动物的头。 </li><li>用手术钳头，背表面上去除皮肤暴露的头骨。 </li><li>用手术刀并没有。 11刀，颅骨额窦的沿内外侧轴的得分，也沿矢状缝在rostrocaudal方向。 </li><li>没有远离头部顶骨去皮。 7弯曲的镊子，小心不要刺破脑组织。 </li><li>将下方的小脑和大脑开始走向嗅球薄锅铲下。用钳子按住头骨的地方，轻轻一拉从头骨的脑，并立即将其放置在冰冷的人工脑脊液（见下面的食谱）。 </li><li>在解剖显微镜下，用消毒解剖剪刀和镊子把大脑中的一半沿中线。旋转，使每个半球的内侧表面（切段）朝上。 </li><li>选择一个半球，内侧面朝上，找到胼胝体压部，胼胝体（胼胝体后区）。 </li><li>做一个切口从皮层的表面沿背腹轴的胼胝体的胼胝体压。 </li><li>去皮，切开皮层向嗅球暴露侧脑室壁的内侧和外侧。 </li><li>旋转，这样的半球背表面朝上，并使用弯曲microscissors，切出，收集暴露的内侧和外侧的墙壁，其中包含的脑室周围地区的NPC位于。 </li><li>重复步骤1.8-1.11另一半球。 </li><li>用移液管的离体组织到7毫升的胰蛋白酶溶液（见下面的配方），在15毫升管中，并放置在37℃的管上的摇杆培养箱中培养25分钟。 </li><li>管在1,500 rpm离心5分钟，吸出上清液，并重新悬浮在2毫升的胰蛋白酶抑制剂溶液（见下面的配方）的组织。 </li><li>轻轻捣碎组织一个小孔径巴斯德吸管30-50倍小心，以避免气泡。 </li><li>离心管在1,500 rpm离心5分钟，吸出上清液，并重新悬浮在1-2ml的SFM（见配方下面）通过捣碎沉淀3-5倍。 </li><li>离心管在1,500 rpm离心3分钟，吸出上清液，重悬在1ml的SFM + EFH。 </li><li>用血细胞计数板在T25培养瓶中的细胞的密度为10个细胞每微升SFM + EFH中，计数活细胞密度。 </li><li>允许的文化成长为7天不受干扰产生的NPC组成的自由浮动的主要神经球。 </li></ol> 2。趋电池准备<ol><li>放置3个方形玻璃。 1盖玻片（22×22×0.17毫米）呐瓶的6N盐酸过夜。 </li><li>第二天，使用金刚石尖端玻璃刀片削减6矩形条（22×5×0.17毫米）的玻璃从方无。 1盖玻片上。 </li><li>酸洗方形单和矩形单传送到在层流罩中。矩形和正方形带先用70％乙醇洗净，然后用组织培养级的灭菌水，并允许上的金擦拭干燥（用于加入无菌，玻璃可能被允许空气干燥）。 </li><li>应用真空脂玻片的平方的一个表面的周边，将它们密封的60毫米的塑料陪替氏培养皿的基础上。 </li><li>应用真空润滑脂沿长轴的长方形玻璃条的一个表面，将它们密封的相对边缘的方形的载玻片（使得它们彼此平行地），以建立一个中部槽。 </li><li>紫外线消毒室，层流罩至少15分钟。 </li><li>移液器250-300μl的聚-L-LYSI东北到中央槽室，室温孵育2小时。 </li><li>约15分钟之前结束的潜伏期，准备Matrigel的解决方案（见下面的配方）。 </li><li>吸取聚-L-赖氨酸，用1毫升灭菌水，和移液管250-300微升到中央槽的Matrigel溶液洗中央槽。 </li><li>在37°C孵育室1小时。 </li><li>吸取基质胶溶液，轻轻地洗1-2毫升SFM中央槽。 </li><li>吸取100微升EFH-SFM或FBS-SFM到中央槽和转移的趋电室搬上舞台，显微镜计数。 </li><li>移液器3-4毫升的神经球的含培养一个60毫米的培养皿中，并转移到陪替氏培养皿的计数显微镜的阶段。 </li><li>在观看目标的5x，使用P10吸管5-8整个神经球（最多四个在一个时间）传输到各趋电槽中央室无解离，并小心地围绕中部槽不破坏基底膜基板的情况下的神经球。 </li><li> EFH-SFM或FBS-SFM添加一个额外的150-200微升到中央的低谷。 </li><li>趋电室转移到在37℃，5％CO 2，100％湿度的培养箱中培养17-20小时（如果分析未分化的NPC）允许神经球坚持的Matrigel的衬底，并分离成单个细胞，如在图1中所示。如果分析差异化的NPC，潜伏期延长到69-72小时，让细胞分化。 </li></ol> 3。活细胞延时成像<ol><li>允许活细胞成像系统平衡，在37℃，5％CO 2中的至少30分钟的时间推移记录开始前。 </li><li>剪下的两个12厘米的块1毫米直径的银线，线圈的一端，并将其放置在高乐氏（Clorox）b浸20分钟，以形成Ag / AgCl电极。 </li><li>转移的趋电性室的计数显微镜搬上舞台，并选择该室将被用于活细胞成像迁移分析基于以下标准：i）本神经球应该是几乎完全离解成单个细胞及ii）应具备的细胞小到没有任何进程从细胞体延伸的圆形形态。 </li><li>转移到在层流罩中选定的趋电室，以及一个单独的正方形无。 1玻璃盖片和真空润滑脂。 </li><li>洗盖滑先用70％的乙醇，然后用无菌水，并应用真空润滑脂在两条平行的盖玻片的边缘的带状。 </li><li>吸出培养基从中部槽室，室的润滑脂条剩下的两个平行的长方形玻璃条，有效地创建一个屋顶上，然后迅速将的盖玻片（油脂面朝下）会议厅。 </li><li>吸取100微升新鲜EFH-SFM或FBS-SFM到中央槽通过毛细作用。 </li><li>使用真空脂培养基池创建边界的中心槽的每一端上，如在图2中示出。 </li><li>剪切PVC管的两个15厘米的片，并使用10毫升的用18号针头的注射器仔细的琼脂糖溶液注入到管中，确保无气泡在管中形成的，使凝胶固化5分钟。 </li><li>转移趋电室的活细胞成像系统中，沿与琼脂糖凝胶管，的Ag / AgCl电极，以及一对空的60毫米的培养皿中，将被用作培养介质储层并且将包含Ag / AgCl电极。允许的趋电室休息内的37℃，5％CO 2的环境，为20-30分钟。 </li><li>在此期间，准备钻孔的2个空培养皿的盖子和趋电性室的皮氏培养皿的盖子到他们具有的Dremel或类似的工具，如在图3中所示。 </li><li>用移液管1-1.5毫升EFH-SFM或FBS-SFM到两侧的中部槽，和7-8毫升的SFM到每一个空的培养皿中。将一个培养皿每一侧的趋电性室的的槽中央和地方，一个Ag / AgCl电极为每一道菜。趋电室和培养皿建立电连续性与琼脂糖凝胶桥桥之间的间隙，如在图4中示出。 </li><li> Ag / AgCl电极连接到一个外部电源，用电流表串联测量电流，并开启电源。使用电压表测量直接跨接在槽中央的电场强度，调整输出的电源，直到所需的电场强度达到（在该实验室进行的检测采用DCEF强度在250 mV /月之间的电流1和1.5毫安）。 </li><li>创议特的时间推移模块上的活细胞成像系统，并允许在实验过程中运行所需的时间量。测定完成后，进行细胞固定在4％多聚甲醛的标准免疫染色分析。 </li></ol>

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没有利益冲突的声明。

该协议已经进行了调整，从以前的研究7-9的行之有效的方法。可以使用各种不同的技术，包括建造一个单独的玻璃以及禁闭细胞接种中部槽10,11的微细化，或使用CO 2激光烧蚀构造galvanotactic室。有些技术可能比别人更费力，代价高昂的。我们描述了一个简单和成本效益的分析，为构建全国人民代表大会趋电性室使用的材料通常存在于大多数细胞生物学实验室。我们的协议，...

<table border="1"><tbody><tr><td height="44"> 项目名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td></td><td></td><td></td><td> 神经前体细胞分离 </td></tr><tr><td> 2M的NaCl </td><td>西格玛</td><td> S5886 </td><td> 11.688克溶解在100毫升蒸馏水2 O </td></tr><tr><td> 1M氯化钾</td><td>西格玛</td><td> P5405 </td><td> 7.456克溶解在100毫升蒸馏水2 O </td></tr><tr><td> 1M MgCl 2的 </td><td>西格玛</td><td> M2393 </td><td> 20.33克溶解在100毫升蒸馏水2 O </td></tr><tr><td> 155毫米碳酸氢钠 </td><td>西格玛</td><td> S5761 </td><td> 1.302克溶解在100毫升蒸馏水2 O </td></tr><tr><td> 0.5M葡萄糖</td><td>西格玛</td><td> G6152 </td><td>溶解在1009.01克毫升蒸馏水2 O </td></tr><tr><td> 108毫米氯化钙 </td><td>西格玛</td><td> C7902 </td><td> 1.59克溶解在100毫升蒸馏水2 O </td></tr><tr><td>青霉素，链霉素</td><td> Gibco公司</td><td> 15070 </td><td></td></tr><tr><td>牛胰腺胰蛋白酶</td><td>西格玛</td><td> T1005 </td><td></td></tr><tr><td>羊睾丸透明质酸酶</td><td>西格玛</td><td> H6254 </td><td></td></tr><tr><td>犬尿喹啉酸</td><td>西格玛</td><td> K3375 </td><td></td></tr><tr><td>卵类粘蛋白胰蛋白酶抑制剂</td><td>沃辛顿</td><td> LS003086 </td><td></td></tr><tr><td> DMEM </td><td> Invitrogen公司</td><td> 12100046 </td><td></td></tr><tr><td> F12 </td><td> Invitrogen公司</td><td> 21700075 </td><td></td></tr><tr><td> 30％葡萄糖</td><td>西格玛</td><td> G6152 </td><td></td></tr><tr><td> 7.5％的NaHCO 3 </td><td>西格玛</td><td> S5761 </td><td></td></tr><tr><td> 1M HEPES </td><td>西格玛</td><td> H3375 </td><td> 23.83克溶解在100毫升蒸馏水2 O </td></tr><tr><td> L-谷氨酰胺</td><td> Gibco公司</td><td> 25030 </td><td></td></tr><tr><td> EGF </td><td> Invitrogen公司</td><td> PMG8041 </td><td>复溶在1毫升的激素组合和等分成20微升单位。 </td></tr><tr><td>成纤维细胞生长因子</td><td> Invitrogen公司</td><td> PHG0226 </td><td>复溶在0.5毫升的激素组合和等分成20微升单位。 </td></tr><tr><td>肝素</td><td>西格玛</td><td> H3149 </td><td></td></tr><tr><td> APO转</td><td> R＆D Systems公司</td><td> 3188-AT </td><td> 0.1 g溶于4毫升蒸馏水2 0 </td></tr><tr><td>腐胺</td><td>西格玛</td><td> P7505 </td><td>将9.61毫克到APO转，所以lution </td></tr><tr><td>胰岛素</td><td>西格玛</td><td> I5500 </td><td> 25毫克，溶于0.1N盐酸0.5毫升增加至3.5毫升卫生署2 0 </td></tr><tr><td>硒</td><td>西格玛</td><td> S9133 </td><td></td></tr><tr><td>黄体激素</td><td>西格玛</td><td> P6149 </td><td></td></tr><tr><td>标准的解剖工具</td><td>精细科学工具</td><td></td><td></td></tr><tr><td>解剖显微镜</td><td>蔡司</td><td> STEMI 2000 </td><td></td></tr><tr><td></td><td></td><td></td><td> 趋电池准备 </td></tr><tr><td>方形玻璃盖幻灯片</td><td> VWR </td><td> 16004 </td><td></td></tr><tr><td> 6N盐酸</td><td> VWR </td><td> BDH3204-1 </td><td></td></tr><tr><td>高真空润滑脂</td><td>道康宁</td><td></td><td></td></tr><tr><td> 60毫米陪替氏培养皿</td><td> Fisher Scientific则</td><td> 0875713A </td><td></td></tr><tr><td>聚-L-赖氨酸</td><td>西格玛</td><td> P4707 </td><td></td></tr><tr><td>基底膜</td><td> BD Biosciences公司</td><td> 354234 </td><td>解冻后取到150微升单位</td></tr><tr><td> FBS </td><td> Invitrogen公司</td><td> 10082139 </td><td>只有使用诱导NPC分化，以其他方式使用SFM + EFH文化传媒如上文所述的</td></tr><tr><td>计数显微镜</td><td>奥林巴斯</td><td> CKX41 </td><td></td></tr><tr><td></td><td></td><td></td><td> 活细胞延时成像 </td></tr><tr><td>银线</td><td>阿法埃莎</td><td> 11434 </td><td></td></tr><tr><td>超纯琼脂糖</td><td> Invitrogen公司</td><td> 15510-027 </td><td></td></tr><tr><td>热灭活ated FBS </td><td>西格玛</td><td> 16140071 </td><td></td></tr><tr><td> PVC管</td><td> Fisher Scientific则</td><td> 80000006 </td><td> 3/32“ID×5/32”OD </td></tr><tr><td>漂白</td><td>高乐氏（Clorox） </td><td></td><td></td></tr><tr><td> 10毫升注射器</td><td> BD </td><td> 309604 </td><td></td></tr><tr><td> 18号针</td><td> BD </td><td> 305195 </td><td></td></tr><tr><td> DREMEL钻</td><td> DREMEL </td><td>型号750 </td><td></td></tr><tr><td>倒置显微镜配备了加湿，孵化室</td><td>蔡司</td><td> Axiovert-200M </td><td></td></tr><tr><td></td><td></td><td></td><td>食谱<table border="1"><tbody><tr><td> 项目 </td><td> 量 </td></tr><tr><td> 2M的NaCl </td><td> 6.2毫升</td></tr><tr><td>1M氯化钾</td><td> 0.5毫升</td></tr><tr><td> 1M MgCl 2的 </td><td> 0.32毫升</td></tr><tr><td> 155MM 碳酸氢钠 </td><td> 16.9毫升</td></tr><tr><td> 1M葡萄糖</td><td> 1毫升</td></tr><tr><td> 108毫米氯化钙 </td><td> 0.09256毫升</td></tr><tr><td>青霉素，链霉素</td><td> 1毫升</td></tr><tr><td>无菌水</td><td> 74毫升</td></tr></tbody></table> 人工脑脊液 <table border="1"><tbody><tr><td> 项目 </td><td> 体积或质量 </td></tr><tr><td>人工脑脊液</td><td> 30毫升</td></tr><tr><td>牛胰腺胰蛋白酶</td><td> 40毫克</td></tr><tr><td>羊睾丸透明质酸酶</td><td> 22.8毫克</td></tr><tr><td>犬尿喹啉酸</td><td> 5毫克</td></tr></tbody></table> &lt;STRONG&gt;胰蛋白酶溶液<table border="1"><tbody><tr><td> 项目 </td><td> 体积或质量 </td></tr><tr><td> SFM </td><td> 15毫升</td></tr><tr><td>卵类粘蛋白胰蛋白酶抑制剂</td><td> 10毫克</td></tr></tbody></table> 胰蛋白酶抑制剂解决方案 <table border="1"><tbody><tr><td> 项目 </td><td> 量 </td></tr><tr><td>无菌水</td><td> 37毫升</td></tr><tr><td> 10X DMEM/F12 </td><td> 10毫升</td></tr><tr><td> 30％葡萄糖</td><td> 2毫升</td></tr><tr><td> 7.5％的NaHCO 3 </td><td> 1.5毫升</td></tr><tr><td> 1M HEPES </td><td> 0.5毫升</td></tr><tr><td>转铁蛋白，腐胺溶液</td><td> 4毫升</td></tr><tr><td> 25毫克胰岛素的解决方案</td><td> 4毫升</td></tr><tr><td>硒</td><td> 100亩; L </td></tr><tr><td>黄体激素</td><td> 100微升</td></tr></tbody></table> 激素组合 （100毫升总，储存在-20°C） <table border="1"><tbody><tr><td> 项目 </td><td> 量 </td></tr><tr><td>无菌水</td><td> 37.5毫升</td></tr><tr><td> 10X的DMEM/F12（3:1） </td><td> 5毫升</td></tr><tr><td> 30％葡萄糖</td><td> 1毫升</td></tr><tr><td> 7.5％的NaHCO 3 </td><td> 0.75毫升</td></tr><tr><td> 1M HEPES </td><td> 0.25毫升</td></tr><tr><td>激素组合</td><td> 5毫升</td></tr><tr><td> L-谷氨酰胺</td><td> 0.5毫升</td></tr><tr><td>青霉素，链霉素</td><td> 0.5毫升</td></tr></tbody></table> EFH-SFM无血清培养基：添加10μl的表皮生长因子，10μl的成纤维细胞生长因子，肝素FBS-SFM和3.66微升：添加0.5毫升FBS <ta表边境=“1”&gt; <tbody><tr><td> 项目 </td><td> 量 </td></tr><tr><td>基底膜</td><td> 150微升</td></tr><tr><td> SFM </td><td> 3.6毫升</td></tr></tbody></table>应Matrigel的解决方案 Matrigel的等分试样放置在一个盒子里的冰和允许缓慢解冻超过4-5小时，以形成一种粘稠的液体混合之前与SFM。这将确保形成一层光滑的Matrigel基板。如果不解冻缓慢，导致基板包含一丛丛的Matrigel，可能会阻碍细胞的迁移。 <table border="1"><tbody><tr><td> 项目 </td><td> 体积或质量 </td></tr><tr><td>超纯琼脂糖</td><td>在10毫升DDH 2 O 300毫克</td></tr><tr><td> SFM 热灭活FBS </td><td> 8毫升 2毫升</td></tr></tbody></table> 基底膜解决方案MIX 8毫升SFM 2毫升热灭活FBS在15毫升猎鹰管。混合琼脂糖用10ml DDH 2 0的埃伦迈尔烧瓶中，并在10秒的间隔在30秒的微波的热量，以确保从微波炉中取出该溶液后，每10秒时间间隔，并彻底混合。最后10秒的微波期，混合的的琼脂糖溶液与SFM / FBS的解决方案和存储在57℃水浴。 </td></tr></tbody></table>

a galvanotaxis assay for analysis of neural precursor cell migration kinetics in an externally applied direct current electric field

神经干细胞和祖细胞（统称为神经前体细胞）（的NPC）在成年哺乳动物大脑的发现已导致一个机构的研究，目的是在利用这些细胞的多能和增殖特性的发展的神经再生策略。这种战略成功的一个关键步骤是动员的NPC外源性移植后向病变部位或提高内源性的前体，发现脑室周围白质区域的中枢神经系统的反应。因此，有必要了解的机制，促进，引导，增强人大迁移。我们的工作主要集中在利用直流电场（量增多），以促进和直接的NPC迁移 - 这种现象被称为趋电性。内源性生理电场的正常发育和伤口修复细胞迁移过程中发挥作用的关键线索。药理中断在蝾螈胚胎的的跨神经管潜力会导致严重的发育畸形。的速度修复受伤的角膜伤口愈合的情况下，直接与上皮损伤后产生的伤害潜力的幅度，药理增强或中断，这DCEF 2-3所示。我们已经证明，成年室管膜下的NPC进行快速和导演的阴极迁移的影响 ，当暴露在外部施加的dcEF。在这个协议中，我们将介绍创建一个简单而有效的高分辨率，长期观察的细胞体易位（迁移）在单细胞水平的趋电检测实验室的技术。该测定中，将适合于调查机制调节dcEF转导到细胞运动通过使用转基因或基因敲除小鼠，短干扰RNA，或特定的受体激动剂/拮抗剂。

运动学分析表明，在250毫伏/毫米dcEF的存在下，未分化的NPC表现出高度定向和快速的趋电向阴极（ 图5A，电影1）。在一个dcEF缺席，随机运动的细胞中观察到（ 图5B，电影2）。在此输入强度，&gt; 98％的未分化的NPC迁移整个6-8小时，这是他们的成像，并自死细胞不迁移，这表明，它们在此期间，在存在或不存在的dcEF仍然可行。 有区别的表型进行可以忽...

Watch this Scientific Journal Video about A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field at JoVE.com

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

在这个协议中，我们将演示如何构建自定义室，允许应用程序的直流电场，使时间的推移成像的成人脑源性神经前体细胞转趋电性。

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-galvanotaxis-assay-for-analysis-neural-precursor-cell-migration

Research

JoVE Journal

Neuroscience

12.4K 次观看.  University of Toronto. 在这个协议中，我们将演示如何构建自定义室，允许应用程序的直流电场，使时间的推移成像的成人脑源性神经前体细胞转趋电性。

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

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). ...

科研

神经科学

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

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

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 ...

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 ...

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 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 ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

自由运动大鼠皮质局部电位、心电图、肌电图和呼吸节律的同步记录

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

We introduce a reproducible and stable optical recording method for brain slices using voltage-sensitive dye. The article describes voltage-sensitive dye staining and recording of optical signals using conventional hippocampal slice preparations.

使用电压敏感染料在脑切片中进行宽场单光子光记录

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation.
In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity.
Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs.
This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

We describe the production of mixed cultures of astrocytes and oligodendrocyte precursor cells derived from fetal or adult neural stem cells differentiating into mature oligodendrocytes, and in vitro modeling of noxious stimuli. The coupling with a cell-based high-content screening technique builds a reliable and robust drug screening system.

胎儿和成体神经干细胞衍生少突胶质细胞前体细胞培养物的高内涵筛选分化与成熟分析

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance ...