Name: enumeration of neural stem cells using clonal assays
Uploaded: 2016-10-04T15:00:00.000+00:00
Duration: 10 min 32 s
Description: Watch this Scientific Journal Video about Enumeration of Neural Stem Cells Using Clonal Assays at JoVE.com

我们感谢该机构科学，技术和研究局（A * STAR），新加坡资助这项研究。

按照IACUC和NACLAR准则（（http://www.brc.astar.edu.sg/index.php?sectionID-11）进行动物的治疗和通过动物部门批准。 注:神经球培养物从/ 6小鼠如前所述5胚胎（E14）的C57BL的前脑制备。 1.克隆样品神经球的培养（1天） <ol><li>由Dulbecco改进的Eagle培养基/养分的F-12（DMEM / F12）培养基有B27补充组合制备NSC生长培养基（最终浓度:1×），表皮生长因子（终浓度:20毫微克/毫升）和bFGF（终浓度10纳克/毫升）和青霉素/链霉素（最终浓度:1×）。 </li><li>游离在1毫升NSC生长培养基和1毫升0.05当量氢氧化钠7分钟在室温E14.5鼠标神经球。吸取细胞上下偶尔保持细胞悬浮。加入1毫升0.05N的盐酸至中性IZE碱。 注意:神经球细胞以20,000个细胞/ mL接种在10cm的培养皿，并且5天培养于37℃，5％ 的 CO 2。预期收益率后每10 cm培养皿约10亿个细胞。然后，这些细胞被离解，并在克隆密度接种在步骤1.2和1.3，分别说明。 <ol><li>使用台盼蓝染色和血球进行细胞计数。 </li></ol></li><li>种子单E14.5小鼠神经球的细胞在96孔培养皿50个细胞/ mL或低于100微升NSC生长培养基的密度。孵育细胞在37℃，5％CO 2的1 w至产生克隆神经球。 注:克隆在此密度神经球形成期间保证作为细胞不相互接触。 </li></ol> 2.为神经球条件培养基培养（3天） <ol><li>离解E14.5小鼠神经球如在步骤1.2中描述。 </li><li>种子的单细胞给E14.5小鼠神经球在在10cm的培养皿10毫升NSC生长培养基中20,000个细胞/毫升的密度。孵育细胞在37℃，5％CO 2的5天，以产生批量培养的神经球。 </li></ol> 3.神经球的制备条件培养基（8日） <ol><li>通过混合700微升制备涂布溶液0.01％聚-L-赖氨酸（PLL）/ 70微升的1毫克毫升层粘连蛋白和磷酸盐缓冲盐水6,230微升（PBS）中。 </li><li>外套10cm的培养皿7毫升的PLL /层粘连蛋白溶液2小时，在37℃。 </li><li>用10毫升血清移液管，所有的散装培养神经球转移到15毫升锥形离心管中。离心神经球在171 xg离心在RT 1分钟，完全除去上清液。 </li><li>和胎牛血清（FBS）（最终浓度:0.5％）的Ad:由DMEM / F12培养基有B27组合（1×最终浓度）制备NSC分化培养基ð10毫升NSC分化培养基，以神经球。移液器上下几次悬浮神经球。 </li><li>抽吸锁相环/层粘连蛋白从10厘米培养皿，并用10毫升PBS洗涤一次。传输所有神经球的带有PLL层粘连蛋白/10厘米培养皿。孵育O / N在37℃，5％ 的 CO 2的神经球坚持菜。 </li></ol> 4.样品测定神经球NFU（8日） <ol><li>计数神经球的数目;形成在各孔，在显微镜下（接种第1天第1部分）。 注:确定每100个细胞铺板形成神经球的数量，这给NFU。 </li></ol> 5.将样品神经球50孔腔室该盖玻片（8日） <ol><li>涂层在37℃的2小时的PLL /层粘连蛋白涂层溶液10μL的50孔腔盖玻片的每个孔中。 注:在说明准备涂液步骤3.1。 </li><li>制备3×50含有各30mL的PBS mL锥形离心管中。使用无菌镊子，取出涂50孔腔盖玻片。浸腔盖玻片到PBS的第一管，接着由第二和随后的PBS第三管，以洗掉的涂布溶液。 </li><li>吸从腔盖玻片的孔中任何残留的液体。 </li><li>轻轻吸取所有介质和神经球从96孔培养皿的每个孔中，并将其转移到一个单一的10厘米培养皿。 </li><li>在显微镜下，挑用装有10微升枪头，一个P10微量样本神经球。确保只有一个单一的神经球是由吸液管列设置为2微升每次拾起。使用新的枪头，每个神经球。 </li><li>传送神经球上涂覆50孔腔盖玻片的孔的中心。 </li><li>重复步骤5.5和5.6，直到盖玻片墓室CONTA的每一个孔INS一个神经球。 注:神经球可放置在层流罩或在工作台上，在显微镜下被拾取。 </li><li> NSC的10微升分化培养基加入到墓室盖玻片每口井。放置在10cm的培养皿和吸管的PBS沿着盘以防止NSC分化培养基干燥的边缘的腔盖玻片。将最多两个腔盖玻片在一个10厘米培养皿。孵育腔盖玻片（包含在10厘米培养皿），在37℃，5％CO 2过夜。 </li></ol> 6.样品分化的神经球（9日） <ol><li>传送从批量培养的神经球（从第3部分），以15毫升锥形离心管中的分化培养基。确保分化培养基的2-3毫升保持在培养皿，以防止粘附的细胞从分离。用20毫升无菌注射器，通过0.2μ通过分化培养基微米的过滤器以除去任何细胞或碎片。 </li><li>传送过滤分化培养基回含粘附细胞的10厘米培养皿。 </li><li>使用无菌镊子，所述腔盖玻片轻轻放置到分化培养基中以反向的方式使得样品神经球都朝下。孵育3天的分化培养基中的腔盖玻片，在37℃，5％ 的 CO 2。 注:样品神经球都与分化培养基接触，但不与下面的分化细胞接触。 </li></ol> 7.固定分化的神经球（12日） <ol><li>轻轻地取出使用无菌镊子的10厘米培养皿中腔盖玻片。轻轻沾盖玻片到PBS洗掉的条件培养基。 </li><li>剥去盖玻片的硅胶垫片。轻轻地，慢慢地执行此，以避免破坏盖玻片。搭边的记下日Ë分化神经球得到遵守。 </li><li> 4％低聚甲醛（PFA）的吸移管1毫升到一张石蜡薄膜切割到盖玻片的大小。轻轻地放在同与煤灰接触向下朝向神经球的石蜡膜盖玻片。修复神经球在室温20分钟。 注意:PFA是有毒的既在液体和蒸气形式，并且可燃。避免吸入蒸气和接触皮肤和眼睛。在通风橱务必抓住PFA。 </li><li> PBS的吸取1毫升到一块石蜡膜。轻轻放置盖玻片朝下接触的神经球用PBS在室温下10分钟洗神经球。 </li><li>重复步骤7.4两次。 </li></ol> 8.分化的神经球O4染色（12日） <ol><li>制备在PBS中的3％牛血清白蛋白（BSA）。吸管将1mL的3％BSA到一张石蜡膜构成。轻轻placin阻断神经球克盖玻片（与神经球朝下）与牛血清白蛋白在室温30分钟的接触。 </li><li>小鼠抗O4的IgM抗体的吸移管1毫升（1:300稀释在3％BSA）到一张石蜡薄膜，并放置于与在与抗体接触向下朝向神经球的石蜡膜盖玻片。允许发生在室温2小时O4染色。 </li><li>如步骤7.4描述的PBS洗盖玻片两次。 </li><li>绿色荧光染料缀合的抗小鼠IgM抗体的二级抗体的吸移管1毫升（1:500中的3％BSA中稀释）到一张石蜡薄膜，并放置于与在与抗体接触向下朝向神经球的石蜡膜盖玻片。留在室温在黑暗中1小时。 注:这一步开始在黑暗环境中进行染色。 </li><li>如步骤7.4描述的PBS洗盖玻片两次。 </li></ol> 9. TUJ1和胶质纤维酸性蛋白（GFAP）染色分化的神经球（12日） <ol><li>准备加入的Triton-X 100（最终浓度:0.5％）透化溶液的3％BSA中。吸取1毫升透液到一张石蜡膜的。放置盖玻片上以与在RT 20分钟的透化溶液接触向下朝向神经球的石蜡膜。 </li><li>如步骤7.4描述的PBS洗盖玻片两次。 </li><li>小鼠的吸移管1 mL抗TUJ1抗体2A抗体（1:在3％的BSA 1:500稀释）和兔抗GFAP IgG抗体（1:3％BSA 1000稀释）到一张石蜡膜和地点盖玻片上在与抗体接触向下朝向神经球的石蜡膜。允许TUJ1和GFAP染色发生在室温2小时。 注意:两个抗体可以作为一个单一的溶液来制备。 </li><li>如步骤7.4描述的PBS洗盖玻片两次。 </li><li>红色荧光染料吸取1毫升共轭的抗小鼠IgG 2A（1:500稀释在3％BSA）和远红色荧光染料缀合的抗兔IgG（1:500稀释在3％BSA）中的二抗到一张石蜡膜和地点盖玻片上以与所述抗体的接触向下朝向神经球的石蜡膜。留在室温1小时。 注意:两个抗体可以作为一个单一的溶液来制备。 </li><li>如步骤7.4描述的PBS洗盖玻片两次。 </li><li>稀4&#39;，6-二脒基-2-苯基吲哚（DAPI）原液在PBS 300纳米。 DAPI的吸移管1毫升到与向下朝向接触用DAPI在RT 2分钟，细胞核染色的神经球的石蜡膜一片石蜡膜和地点盖玻片。 </li><li>如步骤7.4中所述使用去离子水冲洗盖玻片一次用PBS两次。安装在使用合适的安装介质载玻片的盖玻片并晾干O / N在室温。 </li></ol> 10.伊玛分化的神经球更改（13日） <ol><li>使用40X共聚焦显微镜下的图像神经球。使用激光器在488nm，594 nm和647 nm到检测少突胶质细胞，神经元和星形胶质细胞，分别。 </li><li>确定单能，双潜能和立方幂神经球的百分比。 注: -少突胶质细胞，神经元和星形胶质细胞5,14,15,26效力是通过神经球的分化成三种神经谱系的能力来确定。少突胶质细胞被染色阳性确定了O4（第8条）。神经元被染色阳性确定了TUJ1（第9条）。星形胶质细胞被染色阳性确定了GFAP（第9条）。一单能神经球分化成只有血统之一，对于任何一个O4，GFAP或TUJ1应积极染色。一个双潜能的神经球分化成任何两个谱系和O4和GFAP或O4和TUJ1或GFAP和TUJ1应积极染色。一立方幂等不同的神经球iates到所有三个谱系和O4，GFAP和TUJ1应积极染色。 </li><li>确定在通过使用以下公式兴趣群体NSC频率: NSC频率=（立方幂神经球NFU X％）/ 100％。 </li></ol>

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The authors declare no competing financial interests.

我们描述熙神经干细胞的克隆条件下枚举。关键的步骤包括使用新鲜NSC生长和分化培养基中，也使用新鲜制备的PLL /层粘连蛋白溶液的涂覆腔盖玻片，以及培养皿。这保证了神经球的最佳生长和分化的条件。使用质量良好的抗体有效地检测的神经元和神经胶质细胞，以及克隆神经球是不与其它神经球接触的选择性采摘，是至关重要的。此外，重要的是要确保所拾取神经球不涸。建议采摘每10神经球后分化培养基的10微升添加至各孔中。使用一个腔盖玻片在每采样1 10cm皿，以避免来自不同样品的神经球之间的串扰是重要的。如果两个盖玻片（来自不同样品各自含有神经球）被放置在相同10cm皿，从一个盖玻片神经球可能释放可能会改变在其他盖玻片神经球的倾向分化成特定细胞谱系的因素。在相同的10cm平皿两个盖玻片也可导致样品间混合起来。 在事件的NFU或NSC频率低于预期，确保低传代次数神经球被使用。同样重要的是，健康的低传代神经球用于生成条件培养基。接着，如果神经球是与小的直径，如在96孔培养皿的孔中培养，那么这将是困难的操纵和使用微量挑神经球。在这种情况下，神经球转移到一个较大的培养皿，例如6孔皿，采摘前。一些捡到神经球会的文化和染色过程中从墓室盖玻片升空。最小化的腔室盖玻片培养过程中的移动，以及较不密集的洗涤免疫期间ING，将有助于避免神经球的脱离。 最初的研究只使用NFA 7,11,13量化神经干细胞。然而，NFA高估NSC频率既神经干细胞和早期纳米粒子生成神经球5。路易斯等人开发的另一种方法来量化的NSCs称为NCFCA 21,22。所述NCFCA涉及培养的单个细胞在胶原基基质，以确保克隆，并已表明大于2毫米直径的神经球仅通过神经干细胞形成。类似于NCFCA，克隆是整个协议的保证。这是通过产生在克隆密度神经球，并在腔盖玻片区分神经球来实现的。使用分室盖玻片赋予许多优点。所述腔盖玻片允许每个样品神经球，而不必与其他样品神经球物理接触，从而分化过程中确保克隆分化。该腔盖玻片可以放置暂停分化培养基，使样品神经球被连续空调，以确保最大的差异上。在分化过程中不存在的条件培养基和血清，神经球的生存降低，神经球大多产生星形胶质细胞（谭M和艾哈迈德S，未发表）。此外，该免疫染色神经球可以方便地存放时间长周期和腔盖玻片可直接安装在显微镜载玻片上。此外，免疫染色神经球的成像变得容易作为一个可以快速定位在小腔以及神经球，捕获图像，并移动到下一个孔。 我们已经确定了同时使用在这个手稿27日和28 NCFCA描述的协议在E14.5小鼠神经文化NSC频率。该NSC频率E14.5小鼠神经球由双方确定我thods具有可比性。该NCFCA枚举是多和有长期的自我更新能力的神经干细胞。由于我们的方法NSC频率相媲美，从NCFCA，从我们的协议读出似乎并没有高估NSC频率。该NCFCA需要三个星期才能完成，主要涉及细胞接种和中等补货。这里所描述的协议需要13个天进行，并涉及一个不同系列的步骤， 例如 ，神经球拾取和免疫染色。这个协议可以作为一种替代方法枚举神经干细胞。研究人员可以同时使用NCFCA这个协议来验证他们的样品在NSC频率。 该协议的一个限制是一系列重要步骤必须全部在8天的条件培养基的制备，样品测定神经球NFU的，和样品神经球转移到50孔的腔室盖玻片执行必须在执行DAŸ8.有人可能会需要一段时间才能完成这些步骤。此外，它需要实践来以高效的方式执行这些步骤。 神经球已被广泛用来研究NSC功能和行为。然而，神经球同时包含神经干细胞和NPS的。因此，为了从神经球丰富的神经干细胞，研究NSC生物学是重要的。目前，细胞表面标记物，染料排除属性以及细胞大小和粒度已经用于富集的NSCs 6,7,9-13,29,30。该协议可以用于通过潜在NSC标记量化NSCs的富集，并以促进明确NSC标记搜索。近四年的研究已经用此协议枚举NSC频率5,14,15,26。

神经干细胞（NSCs）是中枢神经系统（CNS），可以自我更新和具有多的细胞。神经干细胞是胚胎前脑的发育过程中第一指定，并继续在特定区域的成人大脑坚持如侧脑室的脑室下区（SVZ）和海马齿状回（DG）。许多NSC行正在临床试验中治疗中风和其他神经系统疾病，如巴腾病1中。 神经干细胞和神经祖细胞（NPS）文化传播浮动称为神经球的三维球体结构。神经球培养体系是由雷诺和Weiss在90年代初开发的，当他们发现胚胎和成年皮层细胞能够在表皮生长因子（EGF）和碱性成纤维细胞生长因子（bFGF）2-4存在分裂。神经球由C的异构混合厄尔在不同发育阶段5包括子类。它是具有挑战性的研究专门使用从神经球获得的数据的神经干细胞，由于纳米粒子的存在。因此，关键的是从神经球丰富的神经干细胞。目前，四种主要方法已被用于富集在体外的神经干细胞。第一种是利用细胞表面标记物。路易斯X（LEX）和CD133（也称为Prominin1）是最突出的细胞表面标记物的NSC 6-9。聚糖-1，缺口-1和整合-β1是丰富的神经干细胞10等表面蛋白。二是染料排斥。它已经显示，侧群细胞，其具有泵出的荧光DNA结合染料赫斯特33342的独特能力，被富集的神经干细胞11。三是使用形态选择。已经证明，与增加的细胞大小和粒度细胞怀有更多的神经干细胞比它们的对应5,12,13。四是增加NSC苏rvival因子至培养基。已经显示，加入硫酸软骨素蛋白多糖和载脂蛋白E增强NSC存活并因此增加NSC频率14,15。虽然许多标记物包括转录因子已与神经干细胞16,17相关联，但没有这些标记都能够以丰富的神经干细胞，以纯度。由于缺乏明确的NSC标记，但仍进行量化的NSCs 和体外从纳米粒区分它们是一个挑战。 最初的研究中使用的神经球形成测定（NFA）来量化的NSCs 7,11,13。在该测定中，解离的细胞进行培养，以形成神经球并为每100个细胞铺板，确定产生的神经球的数量。此值称为神经球形成单位（NFU）。在NFU等于NSC频率，如果所有神经球从神经干细胞产生。然而，结果表明，NFU高估NSC频率神经球被两个NS形成Cs和早期的NP 5。因此，它是不准确的枚举的NSCs完全基于神经球的形成。这可能是可以基于自己的能力，以量化的神经干细胞自我更新，增殖广泛和多能产生神经球。 神经干细胞，这是EGF和bFGF的响应，通常存活至少十个通道，从而显示在培养4,18-20广泛的自我更新能力。纳米粒子，这是EGF和bFGF反应为好，也可以产生用于几段而不是在延长的时间周期神经球。因此，它已被广泛接受的是善意的NSCs可以公平准确性基于神经球的形成为至少十通道被枚举。在大多数研究中，但是，自我更新通常是基于二级或三级神经球形成测定由于十通道所需的长实验时间。因此，次级NFA可用于广泛比较自我更新能力的赌注吐温群体，但不能用来准确地枚举NSC频率。 神经干细胞相比，纳米颗粒有更大的增殖能力。神经干细胞的这种特性用路易斯等人 。制定NSC计数的测定-神经细胞集落形成细胞检测（NCFCA）21,22。在该试验中，单电池是一种用于在含胶原的半固体基质培养3周。在这些培养条件下，它表明，形成上述直径2mm的神经球细胞是立方幂，并且可以自我更新长期。这些细胞被定义为神经干细胞。因此，神经干细胞被有效地从纳米粒子区分。 神经干细胞具有分化为星形胶质细胞，少突胶质细胞和神经元的能力。为神经球的分化，生长因子被去除和血清被加入到培养基中。如果所有三个神经谱系的神经球观察，则发起该神经球的小区i秒的NSC。然而，分化测定具有一定的局限性。首先，用于分化的培养条件可能不是最佳的用于产生所有三种神经谱系的。事实上，在一个单一的神经球的分化过程中，发生显著细胞死亡和大多星形细胞生成发生（谭M和艾哈迈德S，未发表）。第二，有克隆的问题。为神经干细胞，形成和神经球的分化准确列举有克隆的条件下，其中每一个神经球产生从单个小区下进行。散装悬浮培养，聚集发生在细胞和神经球水平23-25。因此，有可能对每个神经球，以从多个小区或神经球，其中神经球计数和多能的评价复杂产生。最近的证据表明，细胞的聚集不以0.5细胞/μL或以下，和低铺板密度发生时培养板neu的过程中不会移动rosphere形成15。因此，在如此低的密度培养细胞将确保克隆能力。 目前，NCFCA是最常用的方法，以从纳米粒子区分神经干细胞和枚举NSC频率。的NCFCA，但是，需要一个相对长的三个星期的时间。在这里，我们描述了一个协议，基于神经干细胞形成神经球多能的能力，以枚举NSC频率。该协议需要只有13天的时间执行。所述NCFCA确保克隆的胶原基质防止神经球的运动。在这个协议中使用的培养条件也允许克隆整个维持。例如，使用50孔腔盖玻片确保了神经球将分化不彼此接触。此外，我们使用的条件培养基是分化过程中提供神经营养因子最大化神经球（ 图1）的分化潜能。

<table><tbody><tr><td>15 mL conical centrifuge tubes&amp;nbsp;</td><td>BD</td><td>352096</td><td></td></tr><tr><td>50 mL conical centrifuge tubes&amp;nbsp;</td><td>BD</td><td>352070</td><td></td></tr><tr><td>20 mL sterile syringe&amp;nbsp;&amp;nbsp;</td><td>BD</td><td>300141</td><td></td></tr><tr><td>50 mL sterile syringe&amp;nbsp;</td><td>BD</td><td>300144</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)&amp;nbsp;</td><td>Biowest</td><td>S1810</td><td></td></tr><tr><td>Mouse anti-Tuj1 IgG&lt;sub&gt;2a&lt;/sub&gt; antibody</td><td>Covance</td><td>MMS-435P-100</td><td></td></tr><tr><td>Rabbit anti-GFAP IgG antibody</td><td>Dako</td><td>Z033401</td><td></td></tr><tr><td>Hemocytometer&amp;nbsp;</td><td>Hausser Scientific</td><td>3100</td><td></td></tr><tr><td>Microscope fitted to laminar flow hood&amp;nbsp;</td><td>Leica</td><td>MZ6</td><td></td></tr><tr><td>Glass slides</td><td>Marienfeld-superior</td><td>1000200</td><td></td></tr><tr><td>Mouse anti-O4 IgM antibody&amp;nbsp;</td><td>Merck Millipore</td><td>MAB345</td><td></td></tr><tr><td>Hydromount</td><td>National Diagnostics</td><td>HS-106</td><td></td></tr><tr><td>Microscope</td><td>Nikon Eclipse</td><td>TS100-F</td><td></td></tr><tr><td>Laminar flow hood</td><td>NuAire</td><td>NU-543</td><td></td></tr><tr><td>96-well culture dishes</td><td>NUNC</td><td>167008</td><td></td></tr><tr><td>10-cm culture dishes&amp;nbsp;</td><td>NUNC</td><td>150350</td><td></td></tr><tr><td>Paraffin film</td><td>Parafilm</td><td>PM999</td><td></td></tr><tr><td>Human Epidermal Growth Factor (EGF)&amp;nbsp;</td><td>Peprotech</td><td>AF-100-15</td><td></td></tr><tr><td>Recombinant human basic Fibroblast Growth Factor (bFGF)&amp;nbsp;</td><td>Peprotech</td><td>100-18B</td><td></td></tr><tr><td>0.2 &amp;micro;m filter&amp;nbsp;</td><td>Sartorius Stedim</td><td>16534-K</td><td></td></tr><tr><td>0.45 &amp;micro;m filter</td><td>Sartorius Stedim</td><td>16555-K</td><td></td></tr><tr><td>1.0 N Sodium Hydroxide (NaOH)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S2770</td><td></td></tr><tr><td>1.0 N Hydrochloric acid (HCl)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H9892</td><td></td></tr><tr><td>Trypan Blue&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T8154</td><td></td></tr><tr><td>Poly-L-Lysine (PLL)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P8920</td><td></td></tr><tr><td>Paraformaldehyde (PFA) powder&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P6148</td><td>Toxic</td></tr><tr><td>Bovine Serum Albumin (BSA) powder&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>50-well chambered coverslips&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C7735</td><td></td></tr><tr><td>Stir plate with heat function&amp;nbsp;</td><td>Stuart</td><td>UC152</td><td></td></tr><tr><td>DMEM/F12 medium</td><td>Thermo Fisher Scientific</td><td>11320-033</td><td></td></tr><tr><td>B27 supplement</td><td>Thermo Fisher Scientific</td><td>17504044</td><td></td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>Laminin</td><td>Thermo Fisher Scientific</td><td>23017-015</td><td></td></tr><tr><td>1x Phosphate Buffered Saline without Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt; (DPBS)</td><td>Thermo Fisher Scientific</td><td>14190144</td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-mouse IgM antibody</td><td>Thermo Fisher Scientific</td><td>A21042</td><td></td></tr><tr><td>Alexa Fluor 594 goat anti-mouse IgG&lt;sub&gt;2a&lt;/sub&gt; antibody</td><td>Thermo Fisher Scientific</td><td>A21135</td><td></td></tr><tr><td>Alexa Fluor 647 goat anti-rabbit IgG antibody</td><td>Thermo Fisher Scientific</td><td>A31573</td><td></td></tr><tr><td>4&amp;rsquo;,6&amp;rsquo;-diamidino-2-phenylindole dihydrochloride (DAPI)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>D3571</td><td></td></tr><tr><td>Cell-culture centrifuge</td><td>Thermo Fisher Scientific</td><td>&amp;nbsp;RT1 75002383</td><td></td></tr><tr><td>Pasteur pipettes</td><td>Thermo Fisher Scientific</td><td>10006021</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td></td><td></td><td></td></tr><tr><td>Ventilated fume-hood</td><td></td><td></td><td></td></tr><tr><td>Aspirator</td><td></td><td></td><td></td></tr><tr><td>Confocal microscope</td><td></td><td></td><td></td></tr><tr><td>Micropipettes</td><td></td><td></td><td></td></tr><tr><td>Micropipette tips</td><td></td><td></td><td></td></tr><tr><td>Plastic pipettes</td><td></td><td></td><td></td></tr><tr><td>Multichannel pipettes</td><td></td><td></td><td></td></tr><tr><td>Glass beaker</td><td></td><td></td><td></td></tr><tr><td>Sterile forceps</td><td></td><td></td><td></td></tr><tr><td>pH meter</td><td></td><td></td><td></td></tr></tbody></table>

enumeration of neural stem cells using clonal assays

神经干细胞（NSCs）有能力自我更新和产生三个主要神经谱系 - 星形胶质细胞，神经元和少突胶质细胞。神经干细胞和神经祖细胞（NPS） 在体外神经球通常培养。该协议详细描述了如何确定克隆的条件下给定的细胞群的NSC频率。该协议与细胞的播种开始于密度，允许克隆神经球的产生。然后神经球转移至分室盖玻片并在条件培养基，将使该神经球的分化潜能克隆的条件下分化。最后，NSC频率是基于神经球的形成和多能的能力进行计算。该协议的工具包括候选NSC标志物的评估，神经干细胞的纯化，并从纳米粒子区分神经干细胞的能力。这种方法需要13天才能完成，这是很多小号horter比目前的方法来枚举NSC频率。

我们已经用这种协议来评估LEX，已知的细胞表面标志物NSC 7，以丰富的神经干细胞的能力。从本研究的数据将被用于说明的NSCs如何使用在协议克隆测定所列举。 E14.5小鼠神经球的细胞用抗LEX抗体孵育。随后，表达LEX（LEX +）和细胞不表达LEX（LEX - ）细胞，在50个细胞/ mL接种通过荧光激活细胞分选（FACS）和左，以产生克隆神经球为1周。时间推移成像显示，在50个细胞中产生的神经球/ mL的是克隆（ 图2）。全国农民联盟的LEX +和Lex -细胞决定的。它表明，LEX +细胞具有相比LEX一个显著更高神经球形成能力-细胞（ 图3g）。随后生成的神经球诱导分化克隆的条件下，随后免疫染色和成像（ 图3a-E）。 LEX +细胞产生显著多立方幂神经球比莱克斯-细胞（ 图3F）。然后，NSC的频率是基于NFU和立方幂神经球（ 图3g）的％计。基于LEX选择，丰富了NSC频率超过14倍，验证LEX作为NSC标记。我们也决定使用此协议27和28 NCFCA鼠标E14.5神经球的NSC频率。这两种方法报告的E14.5小鼠神经球约2％，可比NSC频率。 <img alt="图1" src="/files/ftp_upload/54456/54456fig1.jpg" /> 图 1. 下克隆条件神经球分化协议。克隆的条件下生长样品神经球被分别放置在每一个PLL /层粘连蛋白-Coated 50孔腔盖玻片好，使其附着O / N。在同一天，批量培养的神经球铺在分化培养基中的PLL /层粘连蛋白包被的10cm皿，并使其附着O / N。第二天，空调从散装培养神经球介质过滤，并放回10cm皿。然后样品神经球倒置到条件培养基，并允许区分3-4天28。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54456/54456fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54456/54456fig2.jpg" /> 从E14.5神经球衍生图2. 生长一个克隆神经球的。将细胞解离，并在96孔培养皿，在50个细胞/毫升。通过时间推移成像单个细胞进行了追踪6天。邻图片生长成神经球网元这样的细胞被示出。注意，神经球保持克隆能力。 （A）:0天; （ 二 ）45年1月19日; （ 三 ）2.95.37; （ 四 ）3.32.76; （ 五 ）4.46.53; （ 六 ）5.61.44;其中第一个数字指的是一天，第二个数字指的是一天中的分数，和第三个数字指的是H的分数。放大10倍。比例尺，100微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54456/54456fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54456/54456fig3.jpg" /> 图3.克隆神经球和NSC频率计数的分化。单神经球挑取的差异化在50孔腔盖玻片3天。一立方幂神经球站的形象代表与（A）DAPI INED和免疫染色（B）GFAP，（C）TUJ1和（d）O4。 （e）表示（A）的叠加- （D）。比例尺，65微米。 （ 六 ）由Lex +和Lex生成幂单，双潜能和立方幂神经球的％ -细胞（平均值±SEM; N = 3）。 （G）NSC频率LEX +和Lex -作为NFU的产品与立方幂神经球的％计细胞<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54456/54456fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Enumeration of Neural Stem Cells Using Clonal Assays at JoVE.com

Enumeration of Neural Stem Cells Using Clonal Assays

神经干细胞（NSCs）是指细胞，其能够自我更新和分化为三个神经谱系。在这里，我们描述了一个协议，以确定在使用克隆的条件下神经球的形成和分化的给定的细胞群体NSC频率。

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. ...

enumeration-of-neural-stem-cells-using-clonal-assays

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

8.2K 次观看.  Agency for Science, Technology and Research (A*STAR). 神经干细胞（NSCs）是指细胞，其能够自我更新和分化为三个神经谱系。在这里，我们描述了一个协议，以确定在使用克隆的条件下神经球的形成和分化的给定的细胞群体NSC频率。 

视频: Enumeration of Neural Stem Cells Using Clonal Assays

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

从脑室周围地区的成年大鼠和人类脊髓神经分离干/祖细胞

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

科研

发育生物学

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median survival following diagnosis is less than 15 months with maximal surgical resection, radiation, and temozolomide chemotherapy. The challenges inherent in developing more effective GBM treatments have become increasingly clear, and include its unyielding invasiveness, its resistance to standard treatments, its genetic complexity and molecular adaptability, and subpopulations of GBM cells with phenotypic similarities to normal stem cells, herein referred to as glioblastoma stem cells (GSCs). Because GSCs are required for tumor growth and progression, differentiation-based therapy represents a viable treatment modality for these incurable neoplasms. 
The following protocol describes a collection of procedures to establish a high throughput screening platform aimed at the identification of small molecules that promote GSC astroglial differentiation. At the core of the system is a glial fibrillary acidic protein (GFAP) differentiation reporter-construct. The protocol contains the following general procedures: (1) establishing GSC differentiation reporter lines; (2) testing/validating the relevance of the reporter to GSC self-renewal/clonogenic capacity; and (3) high-capacity flow-cytometry based drug screening. 
The screening platform provides a straightforward and inexpensive approach to identify small molecules that promote GSCs differentiation. Furthermore, utilization of libraries of FDA-approved drugs holds the potential for the identification of agents that can be repurposed more rapidly. Also, therapies that promote cancer stem cell differentiation are expected to work synergistically with current "standard of care" therapies that have been shown to target and eliminate primarily more differentiated cancer cells.

An efficient screening protocol is presented for the identification of small molecules that promote astroglial differentiation in glioblastoma stem cells (GSCs). The assay is based on a stem cell differentiation reporter whereby the expression of the enhanced GFP (eGFP) is driven by the human GFAP promoter.

流动 Cytometry-based 药物筛选系统对促进细胞分化胶质母细胞瘤小分子的鉴定

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

癌症研究

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 ...

神经科学

Counting Human Neural Stem Cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

人类神经干细胞计数

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

生物学

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). ...

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 ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

单细胞指数分型与内皮细胞小生境联合培养对胚胎造血干细胞前体的克隆分析

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

一个鼠标，两种文化：隔离和成人神经的培养干细胞从单个小鼠的两个神经源性区

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

活体影像随访单细胞追踪监测细胞生物学及多神经种群的谱系进展

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...