Name: cell sorting of neural stem and progenitor cells from the adult mouse subventricular zone and live-imaging of their cell cycle dynamics
Uploaded: 2015-09-14T18:00:00.000+00:00
Duration: 9 min 27 s
Description: Watch this Scientific Journal Video about Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics at JoVE.com

我们感谢为C茹贝尔，V诺伊维尔，V Barroca酒店，J Tilliet和动物的设施之外，所有的工作人员;到J Baijer和N Dechamps的细胞分选; O.艾蒂安技术援助，并答Gouret为她提供行政协助。流式细胞仪和细胞分选进行了在红外对抗流式细胞仪共享资源，通过设备赠款DIM茎极，INSERM，ARC基金会，并建立了CEA。这项工作是由法国电力公司（EDF）的资助。 MD有来自La法甲癌症中心和LM的奖学金从区域法兰西岛（DIM生物疗法）。马克 - 安德烈·Mouthon和弗朗索瓦D. Boussin份额共同高级作者。

该协议被设计符合11月24日的欧盟理事会指令，1986（86/609 / EEC），并已通过了动物福利机构委员会（CETEA-CEA DSV IDF）。 1.基本设置之前，文化和电视显微镜<ol><li>使用玻璃底培养板或μ-板共聚焦显微镜录像。为1 - 5×10 3个细胞/孔，使用96孔平板和24孔平板超过5×10 3细胞/孔。 </li><li>在开始实验前至少一天，准备无菌聚-D-赖氨酸（PDL）涂层板用于粘附单层培养。补充足够的PDL（蒸馏水中的10微克/毫升），以每外套的底部，并培育O / N在37℃。除去PDL溶液，并允许所述板期间在层流的至少2小时，在干燥罩之前漂洗三次， 卫生署2 O操作。如果不立即使用，存储所述涂覆板在-20℃。 </li><li>通过混合NSC基础培养基和NSC扩散补编在9制备培养基:1的比例（见材料表），以及2微克/毫升肝素，20纳克/ ml纯化人重组表皮生长因子（EGF），和10ng / ml的人类重组成纤维细胞生长因子2（FGF-2）。预热培养基至37℃，在使用前的水浴。 </li><li>为SVZ解离，制备木瓜蛋白酶溶液:1mg / ml的木瓜蛋白酶（15 UI /毫升）在含有0.2毫克/毫升的L-半胱氨酸，0.2mg / ml的EDTA和0.01毫克/毫升DNA酶I厄尔的余额盐溶液（EBSS）于PBS中。通过经过0.2μm过滤器灭菌的溶液。平衡在使用前将溶液在37℃。 </li><li>以停止酶反应，制备蛋白酶抑制剂溶液（卵类粘蛋白）:含有0.7毫克/毫升的胰蛋白酶抑制剂II型F12培养基:DMEM。过滤使用0.2微米的过滤器的溶液。 </li><li>准备PBS 0.6％葡萄糖溶液，收集的大脑和PBS 0.15％BSA小号olution用于洗涤步骤和抗体染色。 </li><li>准备清扫工具:剪刀，解剖和搭售镊子，手术刀。浸泡其中在70％的乙醇。 </li></ol>收获成年小鼠的2大脑和SVZ Microdissections <ol><li>牺牲成年小鼠FUCCI 23（2至3个月大的和/或12个月大的老化研究）执行根据，以适当的制度准则颈椎脱位。 </li><li>喷用70％的乙醇的小鼠和用锋利的剪刀剪断头。 </li><li>做一个切口，沿头皮揭示的头骨。 </li><li>执行纵向中线切口处开始对用小剪刀的嗅球的颅底。一定要避免损坏底层的大脑与剪刀片。删除与弯钳，露出大脑在颅骨上开的部分。 </li><li>收集脑在含有0.6％葡萄糖的PBS 15毫米陪替氏培养皿。 </li><li> ðissect走嗅球。将脑在其背表面和使用解剖刀通过视交叉使冠部。 </li><li>在解剖显微镜下，定位朝上朝向实验者切割冠状面的脑切片的延髓一部分。 </li><li>解剖SVZ，取出用细弯钳隔膜然后插入的细钳子1尖端插入纹状体紧邻心室和从周围组织分离SVZ。有关详细信息，请参阅Azari 等人。24。将解剖SVZ到含有1ml的PBS-0.6％葡萄糖的培养皿。 </li></ol> 3. SVZ组织解离<ol><li>剁碎解剖SVZ在陪替氏培养皿，直到没有大块留。 </li><li>沿着用PBS-0.6％葡萄糖转移组织糜到15ml管中并离心，在200×g离心5分钟。 </li><li>弃去上清液，并添加1 ml的预热的木瓜蛋白酶（1毫克/毫升，在步骤1.4中制备），在37℃的水浴中补充有0.01毫克/毫升DNA酶I.孵育10分钟。使用1毫升每只小鼠木瓜蛋白酶。 </li><li>离心在200×g离心5分钟，丢弃上清液。 </li><li>添加1毫升预热的卵类粘蛋白（0.7毫克/毫升，在步骤1.5中制备）以停止木瓜蛋白酶活性。机械通过P1000的枪头轻轻吹打向上和向下20倍进一步分解组织糜成单细胞悬液。避免气泡。 </li><li>通过无菌20微米的过滤器在一个新的15ml试管中通过细胞悬浮液。请一定要洗的细胞过滤器，用PBS 0.15％BSA以避免丢失的细胞。 </li><li>离心在200×g离心10分钟，丢弃上清液。重悬的细胞在含0.15％BSA的100μl的PBS中。 </li></ol> 4.免疫荧光染色的细胞分选对于电池使用FUCCI红小鼠（ 图2A），请使用以下排序抗体:CD24藻红蛋白cyanine7结合物的PC7] CD15 / LEX异硫氰酸荧光素[FITC]偶联和Ax647结合EGF配体。 注:LEX + EGFR +细胞和表皮生长因子受体阳性细胞并不丰富的成年SVZ:≈600和1500个细胞/小鼠分别为9。我们建议的2至3只小鼠汇集SVZ细胞有足够的材料。不超过12只小鼠在同一天的工作，以使细胞分选持续时间不超过3小时。请记住，太多的小鼠在同一天的工作会导致增加的细胞分选的持续时间可能导致增加的细胞死亡和/或细胞分化。 <ol><li>执行FACS染色在100μlPBS中0.15％BSA的每只小鼠（或在200μl的一组2至3只小鼠为最佳染色）的。 </li><li>准备对照管。使用补偿珠粒根据制造商以制备单一颜色控制管9; S协议。选择细胞的级分（从一只小鼠是足够提取出的细胞的1/10），并分离其在4个试管以制备1阴性对照管（未标记细胞）和3荧光减一（FMO）对照管。重悬在200μl每管的PBS 0.15％BSA的细胞。 提示:对于LEX-FITC FMO控制，标记细胞CD24-PC7（1:50）和Ax647结合的表皮生长因子配体（1:200）;为CD24-PC7 FMO控制，标记细胞CD15 / LEX-FITC（1:50）和Ax647共轭的EGF配体（1:200）和用于Ax647共轭EGF配体FMO控制，与CD15 / LeX-标记细胞FITC（1:50）和CD24-PC7（1:50）。 </li><li>对于用于细胞分选的管，使用下面的抗体在所指示的稀释在PBS中0.15％的BSA:CD24-PC7（1:50），CD15 / LEX-FITC（1:50）和Ax647共轭的EGF配体（1: 200）。 </li><li>在黑暗中孵育20分钟，在4℃。用1毫升的PBS 0.15％BSA和离心机在200×g离心10分钟洗涤。悬浮细胞在20081，L每脑PBS 0.15％BSA的。保持细胞分选管在冰上并立即进行细胞分选。 </li><li>如果使用FUCCI绿小鼠，使用分离柱之前的细胞分选作为LEX FITC抗体股比FUCCI绿色荧光相同的发射波长分开LEX阳性和LEX阴性级分（图3A，B）。 <ol><li>首先，在100μlPBS中的0.15％的BSA的标记，在4℃在黑暗中的细胞与小鼠抗人LEX抗体（1:50）15分钟。 </li><li>用1毫升的PBS 0.15％BSA和离心机在200×g离心洗细胞10分钟，然后在黑暗中标记细胞与抗小鼠I​​gM微珠（1:10）15分钟，在4℃。 </li><li>用1毫升的PBS 0.15％BSA和离心机在200×g离心洗细胞10分钟。悬浮细胞在500微升PBS 0.15％BSA和倒通过磁场分离柱的细胞图式，如图3℃。用1毫升的PBS 0.15％B洗柱SA获得莱克斯阴性部分。 </li><li>以获得LEX阳性分数，取出来自磁场分离柱和洗脱细胞用2ml PBS中0.15％的BSA。 </li><li>继续CD24-PC7和Ax647结合的EGF配体染色在4.2所示。 </li></ol> </li></ol> 5.细胞分选注:细胞分选在FACS分拣机在40psi用86微米noozle。 520/35纳米（FITC），575 / 26nm（PE），670 / 20nm的（Ax647）和740LP（PC7）:使用下面的过滤器设置荧光收集。补偿是必要的，以防止假阳性信号作为重叠的FUCCI红色荧光的发射光谱和PC7染料之间找到。 <ol><li>以细胞分选前夕，增加一个重要的染料从死细胞活歧视。我们使用HO（见材料表）在2微克/毫升终浓度。通过流式细胞分选机，并选择日运行阴性对照管（未标记的细胞）使用侧向角散射（SSC）和前向散射（FSC）参数（图1A）E细胞。 注:死细胞排除通过选通只的HO-阴性级分（ 图1A&#39;），然后双峰被排除通过选择脉冲宽度阴性部分（图1A&#39;&#39;）。 </li><li>运行在步骤4.2中制备的单色彩控制和调节的光电倍增管（PMT）电压在必要时（即负人口过高和/或阳性细胞关闭刻度）。在软件的补偿窗口进行颜色补偿。 </li><li>运行FMO控制准备在步骤4.2（LEX-FITC FMO控制，CD24-PC7 FMO控制和Ax647结合的EGF配体FMO控制），并绘制分配门（ 图1）。排序将细胞直接入100微升培养基在1.5ml微型管。 </li></ol> 6.准备细胞显微镜<ol><li>板新鲜排序细胞以1:1的密度秒- 3×10 3个细胞/孔于聚-D-赖氨酸包被的96孔μ-板用300μl培养基。 </li><li>之前的视频显微镜，孵育培养板在37℃和5％的CO 2的至少1小时，以允许细胞粘附。 </li></ol> 7.显微镜设置和图像采集<ol><li>在37℃下的CO 2的气氛中的5％上附连到一个倒置的恒温室中的共聚焦激光扫描显微镜系统:执行使用计划的Apo虚电路20×DIC物镜（0.75 NA）的实时成像。 </li><li>定位96孔μ-板的预热和平衡恒温室中，并用一个恒温盖更换盖子。 </li><li>打开NIS-Elements软件，然后单击菜单栏上的"收购/并购控制/ ND收购"来选择延时选项（长，舞台的位置，共聚焦Z-路段，...），"收购/并购控制/钛垫221;选择目标和"收购/并购控制/ A1plus设置"，选择菜单栏中的每个荧光的PMT水平。选择一个文件夹来保存数据文件。 </li><li>使用ND采集窗口，设置中心每个以及舞台的位置，并选择大图片选择7×7平方毫米。这将创建一个围绕每个中心的马赛克图像良好。对于大马赛克图像的重叠设置为5％。拍照每20分钟24小时。选择计划阿婆VC 20X DIC物镜（NA:0.75）中的钛板窗口。 </li><li>在A1plus设置窗口，获取使用高速共振扫描仪图像在512×512像素格式的1.26微米/像素的分辨率。明使用可视化的细胞形状。在FUCCI红小鼠的情况下，激发红色荧光在561 nm和收集用五十零分之五百九十五纳米过滤器。在FUCCI绿小鼠的情况下，激发绿色荧光在488nm和收集用四十分之五百三纳米过滤器。确定最佳的PMT水平，偏移和激光功率对于每个波长。 注意:我们建议使用自动对焦功能的明路，让软件自动对焦在每次收购之前，每个阶段的位置。提示:甲计划的Apo虚电路20×DIC物镜（NA:0.75）中用于其无需油优异的分辨率。其他目标可根据所需的光学分辨率被使用。 </li><li>选择ND采集窗口上的"立即运行"按钮，开始采集。 提示:按照电脑工作1循环，以确保一切都在正常工作。 </li></ol> 8.图像处理和分析<ol><li>数据直接在NIS-Elements软件通过单独跟踪的细胞分析。提示:为了争取时间，使用NIS-Elements软件保存为.AVI格式的每个位置，并分析了电影ImageJ的。 </li><li>在ImageJ的软件，追踪经历至少2个部门（ 即一个单元单个单元格四细胞集落）。裁剪一个小范围内的小区周围，并选择"图像/复制"。 </li><li>在菜单栏中选择"图像/栈/制作蒙太奇"，使一个蒙太奇。指定要被包括在帧图像的大小和蒙太奇保存为.tif文件为最佳分辨率。 </li><li>要计算的第一个SG / M期长（ 图2C，D），选择一个红色的荧光细胞（在G中1），然后设置T = 0（S期将开始，一旦红色荧光关闭）。计数帧的数目，直到细胞分裂来估计对SG 2 / M的长度。 注:计算的时间取决于每个帧之间的时间间隔。 </li><li>计算以下G1期（图2C，D）中，继续跟踪，只是除以细胞。如果分割单元进入G1期，将有CDT1红色荧光蛋白的积累。计算的帧的数量，直至红色荧光关闭阿加在对每个小区。 提示:为g 1日开始的红色荧光，可能是弱所以选择G 1期的发病帧中小区划分了避免近似的。 </li></ol>

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The authors declare that they have no conflict of interest.

本文所述的细胞分拣技术允许静止的NSCs，激活神经干细胞和它们的性质的其后代能够研究和动力学在成人脑9之间可靠歧视。再加上FUCCI技术，其允许在活细胞23的细胞周期进展的可视化，我们开发了一种快速和有效的技术，以跟随对G 1和SG 2 / M期的细胞周期从年轻成年和老年鼠脑细胞。 在这个协议中使用的细胞分拣技术是标记允许五个主要神经源性群体的纯化从SVZ 9的第一验证组合。这也是第一个验证的技术使静态和激活神经干细胞的区别。值得注意的是，该技术不要求使用转基因小鼠本身 ，当适于转基因小鼠模型ス这是必要的CH为FUCCI。从那时起，Codega的等人 10已经使用了GFAP-GFP / CD133 / EGFR三重标记组合来从他们的活化的对应物区分开的静态的神经干细胞，但它不能被适于FUCCI技术，因为它要求使用GFAP-GFP转基因小鼠。密歇根州等 11个国家制定了GLAST / EGFR / CD24三联标记策略共享，在这项研究中9所使用的技术共同作用的结果。事实上，在Daynac 等人已经发现lex和GLAST神经干细胞标志物之 ​​间的高相关性，研究9。然而，在美国密歇根州等人的技术中使用的GLAST抗体偶联藻红蛋白，因此不能适用于与使用FUCCI红小鼠。 还有需要注意排序前SVZ细胞几个重要的技术点。首先，将解离的步骤是非常重要，因为脑组织必须被解离成单细胞，同时保留结构在完整性用于抗体的标记策略的蛋白质。 0.05％胰蛋白酶-EDTA已被证明是非常有效的解离SVZ组织27，但LEX抗原被认为是高度敏感的，因为几乎所有的LEX-FITC免疫荧光丢失了（数据未显示）。因此，木瓜蛋白酶，使用，因为它是更有效的和比对脑组织的其他蛋白酶破坏性较小。此外，无论是LEX也不表皮生长因子受体CD24也受到影响，木瓜蛋白酶处理。应当指出的是，抗体标记被更改，如果木瓜蛋白酶治疗超过15分钟。我们得到的最佳结果与机械分离（吸管上下20倍）相关的10分钟木瓜蛋白酶处理。 聚-D-赖氨酸涂层允许贴壁细胞的培养，并在培养数天形成菌落。现在人们普遍认为 ，在体外实验带有其局限性28,29。我们建议确定只有第一个细胞周期经历至少一个后续分裂细胞停留尽可能接近到体内细胞表型。 值得注意的是，提及的是，Geminin（绿色荧光）和CDT1（红色荧光）的蛋白质用于设计FUCCI 系统 23以前证明是大量由神经祖细胞表达在早期神经发生在小鼠中30和在成人脑组织9,25 。虽然mKO2-hCdt1（一百二十〇分之三十）构建体主要用在本研究遵循与红色荧光G1期，同时使用构建体[m​​KO2-hCdt1（一百二十零分之三十零）和MAG-hGem（110分之1） ]可以设想，以允许细胞周期的主要阶段的可视化（G 1和SG 2 / M）以及对G 1 / S转换23。双彩色成像的主要缺点是荧光的有限相容集，仍然可以用于细胞标记。一个解决方案是使用separati在列。例如，我们已经成功地从贫使用分离柱细胞CD24阳性细胞级分之前分类和活成像，因为我们使用的CD24-PE抗体共享比FUCCI -红色荧光25相同的发射波长。 很少有研究调查成年小鼠SVZ人口的细胞周期的长度。与我们的技术获得的总细胞周期长度接近一个通过Costa等人的 21估计在体外 ，但我们能够首次来区分不同的细胞周期阶段。 在体内研究中，蓬蒂等人 22使用胸苷类似物的掺入，以确定不同SVZ细胞群的增殖动力学。然而，它们既不能进行连续监测的细胞周期也不跟踪细胞在单细胞水平。这可能是因为它表明一个非均质性强存在机智的问题欣给定的SVZ细胞群体22,25。我们在这里描述的体外技术的替代，容易设置，它允许不同的成人神经源性群体的细胞周期阶段的实时成像在单个细胞水平。 了解神经干细胞和祖细胞的细胞周期的调节仍然是新的治疗方法中的老化或脑病症的上下文中的发展是一个挑战。因此我们的协议可具有广泛的应用。在老龄化的背景下，这种技术也被证明有助于了解对神经干细胞分化衰老的作用。事实上，这是可能的，以确定进入分化的神经干/祖细胞作为它们红色荧光强度是鲜明高出 26。最后，它也可能是感兴趣的利用连续实时成像在一个单细胞水平来研究负责谱系亲细胞内在的和外在的过程回归成人神经干细胞。

静态的神经干细胞（NSCs）是源成年神经1和可以转化成它们的增殖"激活"的形式表达EGFR（aNSCs）2。一旦被激活，它们产生短暂扩充细胞（的TAC）3，然后成神经细胞，为了嗅球（OB）通过星形胶质细胞的管迁移，最终分化成神经元4,5。神经干细胞和它们的后代被组织在专门的"利基市场"架构沿侧脑室，牵连因素控制其扩散6万千。 SVZ神经原细胞的分离和纯化是必要的，照亮其扩散的复杂分子的规定，但仍然很长一段时间了挑战，由于缺乏特异性标记物，并适于技术。 使用流量的新方法术已使可能的神经干细胞和兵卫的隔离- [R后代从成人的SVZ 2,7-11。利用干细胞标记法12以及成神经细胞标志物CD24 13和荧光EGF对增殖细胞2标签 EGFR，我们最近开发出一种FACS策略允许五个主要SVZ神经源性人口的净化:静态和激活的神经干细胞，总可捕量，不成熟以及迁移成神经细胞9。在这里，我们详细描述这种细胞分选技术，以及如何一个LEX / EGFR / CD24三重允许首次染色两者的静态和激活的神经干细胞的分离。 虽然神经发生成年时期仍然存在，生产新的神经元在大脑老化14急剧下降。大多数研究同意逐步减少在SGZ和SVZ 15-20增殖祖细胞的数量。其后果是不是无害的年龄有关的下降，神经发生在SVZ引发n的缩减ewborn神经元在老年人的大脑的嗅球，最终导致在嗅觉辨别减值老年小鼠18。阐明神经祖细胞的细胞周期动力学是了解衰老过程中成年神经的演变的基本机制的关键步骤。最近的研究已经调查了细胞周期成人神经干细胞和谱系进展体外 21 和体内22但它们都没有利用了细胞分选技术和基因编码的荧光细胞周期的探针可视化分离的细胞的细胞周期阶段在单细胞水平。 这里，我们描述一个协议，取，其中将细胞在其细胞周期荧光转基因FUCCI小鼠的优势，使1克和SG 2 / M期23之间的区别。这个协议显示神经干细胞和它们的后代的自成人FUCCI英里如何准隔离策结合的时间推移的视频显微镜允许细胞周期动力学的在单细胞水平的研究。

<table><tbody><tr><td>96-well uncoated glass bottom culture plates&amp;nbsp;</td><td>MatTek Corp.</td><td>P96G-1.5-5-F</td><td></td></tr><tr><td>&amp;micro;-Plates 96-well</td><td>Ibidi</td><td>89626</td><td></td></tr><tr><td>Poly-D-Lysine</td><td>Millipore</td><td>A003E</td><td></td></tr><tr><td>NeuroCult NSC Basal Medium</td><td>STEMCELL Technologies</td><td>5700</td><td></td></tr><tr><td>NeuroCult NSC Proliferation Supplement&amp;nbsp;</td><td>STEMCELL Technologies</td><td>5701</td><td></td></tr><tr><td>Heparin</td><td>STEMCELL Technologies</td><td>7980</td><td></td></tr><tr><td>EGF</td><td>Millipore</td><td>GF144</td><td></td></tr><tr><td>FGF-2</td><td>Millipore</td><td>GF003</td><td></td></tr><tr><td>Papain</td><td>Worthington</td><td>LS003119</td><td></td></tr><tr><td>EBSS</td><td>Invitrogen</td><td>24010-043</td><td></td></tr><tr><td>L-cysteine</td><td>Sigma</td><td>C-7352</td><td></td></tr><tr><td>0.5M EDTA, pH 8.0</td><td>Promega</td><td>V4231</td><td></td></tr><tr><td>DNase I</td><td>Sigma</td><td>D5025-15KU</td><td></td></tr><tr><td>Trypsin inhibitor type II</td><td>Sigma</td><td>T9128</td><td>Ovomucoid</td></tr><tr><td>DMEM:F12 medium</td><td>Life Technologies</td><td>31330-038</td><td></td></tr><tr><td>20 &amp;micro;m filter&amp;nbsp;</td><td>BD Medimachine Filcon</td><td>340622</td><td></td></tr><tr><td>Eppendorf microtubes 3810X</td><td>Sigma</td><td>Z606340-1000EA</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A1595</td><td>Bovine serum albumin solution</td></tr><tr><td>CD24 phycoerythrin-cyanine7 conjugate [PC7]</td><td>Life Technologies</td><td>A14776</td><td>Mouse IgM; clone MMA. 1:50</td></tr><tr><td>CD15/LeX fluorescein isothiocyanate [FITC] - conjugated</td><td>BD Biosciences</td><td>332778</td><td>Rat IgG2b; clone M1/69.&amp;nbsp; 1:50</td></tr><tr><td>Mouse anti-human LeX antibody</td><td>BD Pharmingen</td><td>559045</td><td>Mouse IgM; clone MAM.&amp;nbsp; 1:50</td></tr><tr><td>Alexa647 - conjugated EGF ligand</td><td>Life Technologies</td><td>E35351</td><td>1:200; Ax647-conjugated EGF ligand in the text</td></tr><tr><td>CompBeads</td><td>BD Biosciences</td><td>644204</td><td></td></tr><tr><td>MACS LS separation columns&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-042-401</td><td>separation columns (in the manuscript)</td></tr><tr><td>Anti-mouse IgM microbeads&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-047-301</td><td></td></tr><tr><td>Hoechst 33258</td><td>Sigma</td><td>861405</td><td>HO (in the manuscript)</td></tr><tr><td>INFLUX cell sorter</td><td>BD Biosciences</td><td></td><td>FACS sorter (in the text)</td></tr><tr><td>Nikon A1R confocal laser scanning microscope system attached to an inverted ECLIPSE Ti</td><td>Nikon Corp.</td><td></td><td>confocal laser scanning microscope (in the manuscript)</td></tr><tr><td>NIS-Elements AR.4.13.01 64-bit software</td><td>Nikon Corp.</td><td></td><td>NIS-Elements software (in the manuscript)</td></tr><tr><td>Plan Apo VC 20x DIC objective (NA: 0.75)</td><td>Nikon Corp.</td><td></td><td></td></tr><tr><td>ECLIPSE Ti thermostated chamber</td><td>Nikon Corp.</td><td></td><td>thermostated chamber (in the manuscript)</td></tr><tr><td>ImageJ</td><td>RBS</td><td></td><td></td></tr><tr><td>FlowJo</td><td>Tree Star, Ashland, OR</td><td></td><td></td></tr><tr><td>FUCCI mice</td><td>RIKEN BioResource Center, JAPAN</td><td></td><td>Sakaue-Sawano et al. 2008</td></tr></tbody></table>

cell sorting of neural stem and progenitor cells from the adult mouse subventricular zone and live-imaging of their cell cycle dynamics

在侧脑室的脑室下区神经干细胞（NS​​Cs）（SVZ）维持整个生命的嗅觉神经发生在哺乳动物的大脑。他们先后产生短暂扩充细胞（总可捕量）和神经母细胞分化成神经细胞，一旦他们整合嗅球。新兴的荧光激活细胞分选（FACS）技术允许NSCs的隔离以及它们的子代，并开始阐明基因调控网络在成年神经源性龛的光。我们在这里报告一细胞分拣技术，它允许按照从成年SVZ用LEX / EGFR / CD24三重染色区分上述细胞群的细胞周期动力学。然后分离的细胞铺板以贴壁细胞，探讨在由时间推移的视频显微镜详细它们的细胞周期进程。为此，我们使用转基因荧光泛素细胞周期指示符（FUCCI）小鼠中，细胞是红色荧光durin的G 1阶段，由于A G 1特异性红CDT1记者。这种方法最近发现，神经干细胞增殖过程中的老化逐渐延长它的G 1期，从而导致神经损伤。这种方法是很容易转座到其他系统，可能是对脑细胞在脑病理学的上下文中，细胞周期动力学的研究极大的兴趣。

到成年小鼠SVZ干细胞谱系的不同细胞群之间可靠地鉴别的能力是原始研究其功能特性。为了这个目的，我们开发了一个LEX / EGFR / CD24三标记策略，允许从成年SVZ特定细胞群的净化:静态神经干细胞（LEX 亮），激活神经干细胞（LEX + EGFR +），总可捕量（EGFR +）的，不成熟的和迁移的成神经细胞（CD24 +的EGFR +和 CD24 +，分别）9（ 图1）。施加到FUCCI转基因小鼠，所述细胞分拣技术允许的不同神经源性群体在单细胞水平25的细胞周期阶段的实时成像。 FUCCI红小鼠用于按照G 1期与红色荧光使用时间推移的视频显微镜（ 图2A）。 FUCCI红阴性细胞代表该细胞在S-G2 / M期的细胞周期，FUCCI红POS细胞为G 1细胞和FUCCI红光亮细胞被细胞退出或离开细胞周期（图2B）-23,26-。 LEX + EGFR +和从年轻成年（图2C）和中年小鼠（ 图2D）的EGFR +细胞铺板作为聚D-赖氨酸包被的培养板中的贴壁细胞。第一SG / M期被 ​​确定在单细胞一旦红色荧光关闭，直到细胞分裂。如先前所观察到的SG 2 / M期长度表明，无论是在年轻或中年小鼠LEX + EGFR +和EGFR +细胞之间没有差异。然后，我们计算从第一部的下一个G 1期的长度，直到红色荧光关闭。有趣的是，G 1期延长的LEX + EGFR +细胞，但不老化过程中被发现在EGFR +细胞25（图2C，D）。结果，神经球电镀后第5天获得的是在从老年小鼠获得如图2E LEX + EGFR +细胞小。 FUCCI绿小鼠也可用于可视化对SG 2 / M期的绿色荧光（图3A，B），但细胞必须使用分离柱为LEX-FITC抗体预排序共享相同的发射波长比FUCCI-绿色荧光（图3C）。的FUCCI绿色荧光的第一SG 2 / M期的年轻成年LEX + EGFR +和EGFR +细胞的一个例子示于图3D。 <img alt="图1" src="/files/ftp_upload/53247/53247fig1.jpg" /> 图1:通过FACS小区选择的策略（A）的细胞首先根据吨选定采用侧向散射（SSC）与前向散射（FSC）参数继承人形态。有关形态门选择更多详细信息，请参阅Daynac 等 9（A&#39;）使用HO标记死细胞进行标记和排除的选择。（A&#39;&#39;）双峰是使用脉冲宽度参数排除。为了建立分配门，荧光减一（FMO）的CD24-PC7控制（LEX-FITC / FUCCI红/ EGF-Ax647标签; B）和 LEX-FITC（FUCCI红/ EGF-Ax647 / CD24 -PC7标签; B&#39;）中 ，使用（C，C&#39;）然后，分配门在LEX-FITC / FUCCI红连同FMO控制/ EGF-Ax647 / CD24-PC7标记的管进行了测定。总的细胞的平均百分比的栅极内表示。如果选择CD24 +细胞（神经母细胞），要注意排除CD24 亮表达细胞的大门。事实上，CD24 明亮相当于室管膜CELLS 2,9，C&#39;代表CD24阴性细胞（黑方块C语言）。仅LEX + EGFR +和EGFR +（C&#39;）分配门被用于本研究。 <img alt="图2" src="/files/ftp_upload/53247/53247fig2.jpg" /> 图2:细胞周期的使用FUCCI红小鼠实时分析（A）中 FUCCI红细胞周期的示意图，其中细胞SG 2 / M期23（B）的FACS的代表性过程期间G1期红色荧光和无色。 FUCCI-红色荧光的SVZ细胞（C，D）视频显微镜用LEX + EGFR +和EGFR +细胞分选的年轻（C）和中年（D）FUCCI红小鼠允许G 1期与追踪红色荧光，而SG 2 / M期可以推断磨片n个红色荧光关闭。连续图像示与 D中提出了一个时间刻度。菌落（E）的代表性图像（神经球）中得到铺板后5天。比例尺:10微米（C，D），30微米（E） <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53247/53247fig2large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53247/53247fig3.jpg" /> 图3:策略细胞周期的使用FUCCI绿小鼠活分析（A）中 FUCCI绿细胞周期，其中细胞绿色SG期间中G 1期23 2 / M期和无色的示意图（B）的 FACS表示的FUCCI绿荧光:FUCCI绿阴性细胞代表G中的电池 1相的细胞周期而FUCCI格力ÑPOS细胞是在SG / M 23。（C）在为了遵循绿色荧光SG 2 / M期LEX + EGFR +及EGFR +细胞 ，SVZ细胞必须被预排序以分离柱。细胞用抗小鼠LEX抗体和Lex +馏分保留使用抗小鼠微珠的柱（参见协议4.）。然后，LEX +莱克斯-组分被洗脱，并标有EGF-Ax647和CD24-PC7抗体流式细胞分选（D）LEX + EGFR +的视频显微镜和EGFR +细胞分选的年轻FUCCI绿小鼠允许的跟踪。神光2 / M期绿色荧光。连续图像示与 D中提出了一个时间尺度。比例尺:10μm的（D）中 。

Watch this Scientific Journal Video about Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics at JoVE.com

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

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. ...

cell-sorting-neural-stem-progenitor-cells-from-adult-mouse

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

14.1K 次观看.  CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, UMR 967. 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.

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

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

在成年小鼠V-SVZ细胞的神经分析的稳定，高效的基因修饰干细胞自主和非自主的影响

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

科研

发育生物学

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

神经科学

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

急性片的成年小鼠前脑的神经细胞迁移的延时成像

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

P2X7 受体实时活细胞流式细胞仪检测成人神经祖细胞中的钙影响、毛孔形成和吞噬作用

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

分离和成人神经干细胞的培养从鼠标Subcallosal区

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

生活成像的脑皮层细胞使用二维培养系统

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

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 ...

可视化发育中的小鼠大脑中的神经元和神经胶质细胞动力学

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

作者聚焦：探索发育中的大脑中的细胞迁移和基因作用

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

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

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在成年小鼠V-SVZ细胞的神经分析的稳定，高效的基因修饰干细胞自主和非自主的影响

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

急性片的成年小鼠前脑的神经细胞迁移的延时成像

P2X7 受体实时活细胞流式细胞仪检测成人神经祖细胞中的钙影响、毛孔形成和吞噬作用

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

分离和成人神经干细胞的培养从鼠标Subcallosal区

生活成像的脑皮层细胞使用二维培养系统

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

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像