这项研究导致这些结果根据出让协议已收到的资金来自欧盟第七框架计划（FP7/2007-2013）N°323267，来自法国电力公司（EDF）和L&#39;法新社国立德拉RECHERCHE - 桑特 - 环境在等桑特-劬劳（ANR-SEST，Neurorad）。

1，动物规程该协议已经被设计符合欧共体理事会指令11月24日，1986（86/609/EEC），并已通过了动物福利（CETEA-CEA DSV IDF）机构委员会。 <ol><li>注射用EdU /的BrdU和E14.5孕鼠照射（ 图2A）。 <ol><li>制备的EdU的溶液在1毫克/毫升和BrdU在5 mg / ml的PBS中。执行腹腔注射100微升的溶液的EdU使用25号针头成孕鼠（IP）。 1.5小时后，照射小鼠（全身照射），在0戈瑞或2戈瑞（0.6格雷/分钟）。紧接在后，用25号针头进入孕鼠注射（IP）的200微升的BrdU溶液。 </li></ol></li><li>制备胚胎的大脑冠状切片。 <ol><li>在1小时或4小时照射后，通过二氧化碳安乐死的孕鼠。 </li><li>打开腹腔CAVITY超过用剪刀3厘米。通过切片角的两个末端从子宫的其余部分分开的两个子宫角。转移子宫角到含有PBS 0.6％葡萄糖的培养皿中。为了分离胚胎打开子宫角用钳子。删除使用镊子每个胚胎的羊膜囊。 </li><li>解剖胚胎头与钳和4％多聚甲醛（PFA，pH值= 7.4）O / N修复它们浸泡在4°C。 </li><li>在4℃下冲洗胚胎头在PBS中的至少一个夜头可以保持在PBS中数天。 </li><li>使用真空浸润处理器 ，如表1所示在石蜡中嵌入的头。头嵌入也可以根据化学罩在一个65℃的烘箱中的石蜡浴进行乙醇和xylen浴，然后。 </li><li>准备5微米厚的冠状切片，用切片机。 </li><li>安装到多聚赖氨酸包被的载玻片。&lt;/ LI&gt; <li>幻灯片可以保持在室温（RT）下持续数月。 </li></ol></li></ol> 2，教育/的BrdU染色<ol><li>脱蜡和抗原修复<ol><li>通过幻灯片浸泡在甲苯5分钟的3浴Deparaffinize石蜡包埋脑切片。 </li><li>补充水分在2浴室降低乙醇浓度的各溶液中3分钟，如以下:乙醇100％，95％乙醇，70％乙醇和5分钟的H 2 O。幻灯片可以保持在去离子水中几个小时。 </li><li>煮沸的切片中的柠檬酸溶液（10mM，pH为6）10分钟，然后孵育在去离子水中5分钟。 </li></ol></li><li>染色的EdU <ol><li>根据制造商的方案进行的EdU检测。透化的细胞用0.5％的Triton X-100的PBS中为10分钟。 </li><li>通过稀释于去离子水中的10倍的溶液制备1X的EdU缓冲添加剂。此溶液应新鲜制作，并于同达使用年。 </li><li>准备的EdU反应的鸡尾酒，包括的EdU的Alexa Fluor 488叠氮化物。添加的成分如下表2中所列的顺序，否则该反应将不会进行优化是非常重要的。内准备15分钟用埃杜反应鸡尾酒。 </li><li>除去透化缓冲液（步骤2.1.1），然后用PBS洗两次。 </li><li>添加150微升的EdU反应混合物，把盖玻片，并温育在黑暗中在室温下30分钟。 </li><li>删除埃杜反应鸡尾酒，然后用PBS洗一次。 </li></ol></li><li>的BrdU染色<ol><li>制备饱和缓冲液含7.5％山羊血清和PBS中的7.5％的胎牛血清。加入150μl的饱和缓冲液对脑切片，放盖玻片，孵育1小时在室温阻断非特异性抗体结合。 </li><li>取出盖玻片。加入150μl稀释在饱和缓冲液1/300鼠抗BrdU一抗，把盖玻片和孵化的O /n在4°C。 </li><li>取出盖玻片，然后在PBS中洗涤3次。 </li><li>加入150μl在饱和缓冲液中稀释为1/400的山羊抗小鼠Alexa594第二抗体，将一个盖玻片，并孵育1小时，在室温下。 </li><li>取出盖玻片，然后洗一次在PBS。 </li><li>核染色:加150微升4&#39;，6 - 二脒基-2 - 苯基吲哚（DAPI）在1微克/毫升的PBS中，把盖玻片并孵育在室温下2分钟。 </li><li>在安装介质安装滑轨。 </li></ol></li></ol> 3。分析<ol><li>检查脑切片用20X物镜或激光共聚焦显微镜，荧光显微镜下，在3个通道（488纳米，594纳米和UV）获得的图像作为分开的文件。 </li><li>堆栈用Photoshop软件映像。 </li><li>在背内侧脑壁的标准部门分析脑切片。这个扇区是100微米的在其内侧 - 外侧尺寸和被分成10μ18箱（或更多）使用叠加在图像的网格（ 图1），在其径向尺寸米高度先前描述31。 <ol><li>网格对齐诸如第一bin是否在心室表面，其平行于心室边界长轴。然后按数字标记EDU，的BrdU和/或细胞核固缩（对应于凋亡细胞）在每个箱。号位于在当中载有其较大的部分，或者在较低的垃圾桶，当原子核占据了两个垃圾桶内相等面积的bin两个垃圾桶的边界上的核心。 </li></ol></li><li>统计分析。 </li></ol>分析每个胚胎的至少两个皮质切片。对来自不同窝的至少3个胚胎重复实验。结果应以平均值（SEM）的平均值±标准误差。统计分析与GRAPHPAD棱镜采用双因素方差分析和Bonferroni多重比较进行事后检验进行。

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该作者有没有利益冲突。

这里所描述的基础上的EdU 1.5小时掺入实验设计照射前和BrdU的掺入允许照射后立即NSPCs能够激活S期和G2 / M期检查点，但第1小时过程中没有在G1 / S检查点之后的一个示范基因毒性应激在胎鼠大脑。我们进行了在其中的EdU已经注入在不同时间照射和BrdU后，才刚刚1小时小鼠牺牲其他的实验中，让我们特别欣赏进入S期的速度。这些实验表明，NSPC不激活G1 / S期阻滞在体内 22,23基因毒性应激后。 因此，DNA修复和细胞凋亡之间的选择不会发生在G1 / S期检查点提出的问题对中保证基因组稳定性在这些细胞的机制的性质。 该协议是方便，快捷。不同步骤目前没有明显的困难。这种技术是一种可以替代使用氯脱氧尿苷的（CldU）和碘脱氧尿苷（IDU），其已经显示出交叉反应的32。 一，应用广泛的面板可以被考虑，因为该协议是轻松灵活地适应多种生物学问题。变化的例子提出了罗克和合作者23，艾蒂安和合作者22，并在卢梭和合作者24。它可以同时结合免疫荧光检测的几种​​细胞标记物或参与DNA损伤应答例如蛋白质。注意，在此情况下，两个的BrdU及EDU检测可以与商业化的不同荧光基团进行的，以有利于免疫检测的其他标记。 的EdU和BrdU 在 NSPC 体内掺入也可以在小区中使用依流式细胞仪进行进一步的生化分析。最后，日是可以执行的方法来监测NSPC的细胞周期进程中不同类型的突变小鼠或其他类型的遗传毒性应力（比电离辐射等）或影响细胞周期进程的任何其他的治疗。此外，它可以很容易地适用于其他组织中复制。

在胚胎脑发育，在脑室区产生的大脑皮层的投射神经元中，假复层上皮的不同类型的神经干细胞和祖细胞（NS​​PC）所组成的行侧脑室。其中NSPC，径向胶质细胞（RGC），其作为神经干细胞，经过interkinetic核迁移（INM）:它们在心室和心室区的基极限的S-相的表面进行有丝分裂（VZ） 1，2,3。它们可分为两种对称地产生两个RGC或不对称，以生成一个RGC和一个神经元或中间祖细胞（IPC）的4，5。工控机迁移到上覆的增殖层称为脑室下区（SVZ），其中最后一个对称分裂后，便产生两个不成熟的投射神经元6-8。相反，研资局，IPC不进行INM（9审查）。新生成的神经元MIGR吃了径向沿径向纤维通过中间区（IZ）到达其 ​​最终目的地，皮质板（CP）10，8。所有这些事件的最佳时机是正确的皮质发育至关重要。例如从扩散切换到神经祖细胞群分化是由G1期持续时间11，12控制。 G1期的延长，细胞分化从而关联。 基因毒性应激，如电离辐射，严重危害大脑的发育（13审阅）。我们和其他人已经表明，NSPC是极易受辐射诱导的细胞凋亡14，15,16。电离辐射诱导DNA双链断裂是对增殖细胞的最严重的损害。 DNA损伤反应（DDR）的循环细胞的一个重要组成部分是细胞周期检查点在G1 / S期或G2 / M期的转换或S在激活相（内S检测点）17-21。他们阻断细胞周期进程，为DNA损伤修复或消除过于受损细胞的提供时间。因此，细胞的死亡，以及延迟细胞周期进程可能改变大脑发育响应于电离辐射曝光22-24。因此，这是有趣的，开发一种方法，以评估在NSPC细胞周期检查点在被照射的小鼠胚胎脑的活化。 在细胞周期的进展是经常随后使用的胸苷类似物，5 - 溴-2&#39;-脱氧尿苷（BrdU）标记的掺入。在细胞周期中，当DNA被复制的S期的BrdU掺入。使用针对的BrdU抗体，该抗体的允许细胞，是在S期的BrdU的脉冲期间的随后的检测。 一种新颖的胸苷类似物，5 - 乙炔基-2&#39;-脱氧尿苷（EDU）由叠氮化物的荧光检测。不同的方法来检测的EdU和乙<html> RDU不交叉反应的25，使同时检测两个胸腺嘧啶的类似物，它是细胞周期进程的研究是有用的。通常细胞却先脉冲用EdU，然后用脉冲的BrdU，其中两个法团之间的时间持续两小时25，26。在含有的EdU培养基另外的BrdU导致优先掺入的BrdU到DNA用EdU的排除，而同时加入的EdU 25与等摩尔或等摩尔的一半的BrdU向媒体结果中只BrdU掺入27。这简化了通过消除通常须从培养基中取出的第一个标签之前加入第二标记物的洗涤步骤的双标记方法。这也就是， 在体内研究中，在洗涤步骤是不可能的，以确定进入S期或细胞群体的出口处的准确定时的特别的兴趣。 吨"&gt;最近，已开发了在使用的EdU和BrdU 23，24,22胚胎小鼠大脑的双脉冲标记的方法来分析细胞周期进程和NSPC的INM后在子宫内的辐射，而且它已被证明的是，在S期，小鼠细胞首先复制常染色质区域，然后在臂间异染色质28,29,30。有趣的是，聚集在interphasic核不同染色体臂间异染色质形成异染色质灶也被称为chromocenters和DAPI染色不易察觉的亮灶。因此，常染色质和chromocenters的差的EdU和BrdU染色有助于我们分析更精确地NSPC S期的进展。 这种方法允许的明显缺乏G1 / S期检查点在NSCP 22-24，这是相当令人惊讶，因为这个检查站被认为是对基因组稳定性的关键示范。几个EXP基于对的EdU和BrdU脉冲的各种组合erimental设计已经用于分析细胞周期进程中的腹侧和背侧端脑。这里，我们给协议允许NSPC 在 E14.5小鼠胚胎的子宫内的辐照后的急性DNA损伤的前4个小时内的学习的例子。

<table><tbody><tr><td>EdU</td><td>Life Technologies</td><td>A10044</td><td>1 mg/ml</td></tr><tr><td>BrdU</td><td>Life Technologies</td><td>B5002</td><td>5 mg/ml</td></tr><tr><td>IBL 637</td><td>CIS BIO International</td><td></td><td></td></tr><tr><td>Tissu Tek VIP</td><td>Leica</td><td></td><td></td></tr><tr><td>Microtome</td><td>Leica</td><td>RM 2125 RT</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>93443</td><td></td></tr><tr><td>Click-iT EdU Alexa 488 imaging kit</td><td>Life Technologies</td><td>C10083</td><td></td></tr><tr><td>Anti-BrdU</td><td>GE Healthcare</td><td>RPN202</td><td>1/300</td></tr><tr><td>Goat anti mouse-Alex594</td><td>Life Technologies</td><td>A11001</td><td>1/400</td></tr><tr><td>Fluoromount</td><td>SouthernBiotech</td><td>0100-01</td><td></td></tr><tr><td>Polysine slide</td><td>Thermo scientific</td><td>J2800AMNZ</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>PBS</td><td>Life Technologies</td><td>20012-068</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr><tr><td>Microscope BX51</td><td>Olympus</td><td></td><td></td></tr><tr><td>Confocal microscope SPE</td><td>Leica</td><td></td><td></td></tr><tr><td>Prism software</td><td>Graphpad</td><td></td><td>Version 5.0c</td></tr><tr><td>Photoshop software</td><td>Adobe</td><td></td><td></td></tr></tbody></table>

assessing cell cycle progression of neural stem and progenitor cells in the mouse developing brain after genotoxic stress

从不同类型的神经干细胞和祖细胞（NS​​PC），它形成一个假复层上皮衬里胚胎脑的侧脑室脑发育过程中产生的大脑皮层神经元。遗传毒性压力，如电离辐射，对发育中的大脑与NSPC的高灵敏度非常有害的影响。所涉及的细胞和分子机制的阐明依赖于这些特定类型的细胞中，DNA损伤反应，需要一个准确的方法通过细胞周期来确定受损组织NSPC进展的表征。这里显示的基础上的EdU和BrdU对孕鼠和进一步检测在胚胎大脑冠状切片这两个胸腺嘧啶核苷类似物的连续腹腔注射的方法。的EdU和BrdU都在复制细胞的DNA在S期注册成立，并通过两种不同的方法（叠氮化物或检测的特异性抗体，分别），这有利于它们的同时检测。的EdU和BrdU染色，然后在其从心室率在背端脑的标准区域距离的函数确定每个NSPC核。因此，这种双标记技术允许区分细胞，通过细胞周期进展从那些在DNA损伤反应已经激活细胞周期检查点，导致细胞周期阻滞。 实验的一个例子给出，其中的EdU被照射和BrdU前后立即注入和分析的4小时内下照射进行。这个协议提供了NSPC在细胞周期在它们已经被照射的相位的函数的急性DNA损伤反应的精确分析。这种方法是很容易的转座到许多其他系统，以确定在生物体组织上的细胞周期进展的特定治疗的影响。

在图2中描述的实验中，埃杜给药1.5小时照射和BrdU刚照射后前。四种类型的细胞，然后在1或4小时后照射制备的皮质切片区别，根据任一的EdU或的BrdU，双方或无的掺入（ 图2A和2B）。重要的是，既没有的EdU也不BrdU掺入改变辐射诱导的细胞凋亡（数据未示出）的电平。此外，该染色方法允许在S-期DNA复制过程中EDU和BrdU的唯一的检测中，而没有相应的灵敏度来检测与维修甚至照射后相关的DNA的合成。的确，i）两者的EdU也不的BrdU染色，观察到1小时照射后（PI）在皮质板，其中位于有丝分裂后神经元在G0期，和ii）的EdU（+）和/或的BrdU（数+的细胞）未在照射增加相比unirradiated大脑。 埃杜（+）的BrdU（+）核 埃杜（+）的BrdU（+）原子核对应NSPC那是在S期之前和之后0小时PI。因此，它们被照射在S期。 如图3B和3C的常染色质的EdU是（+），并可能还BrdU阳性。他们chromocenters要么的BrdU（+）或的BrdU（ - ）。根据S期完成在处死时如图2B所示，在1小时的PI，埃杜（+）的BrdU（+）细胞核定位于S期垃圾箱（ 即箱4至10，其中NSPC是已知的执行S期31，它们的分布和数量并没有受到照射。这表明辐射没有在这个时候诱导DNA合成一个完整的块。 始终与基底根尖细胞核移植过程中G2期，NSPC的最埃杜（+）的BrdU（+）原子核附近发现心室在未经辐照的伯莱4小时PI纳秒（ 图2C）。这并没有发生在2 Gy的照射大脑中，原子核留在S期垃圾箱，其中许多是细胞凋亡。总之这个数据表明，NSPC照射在S期与延迟INM和细胞死亡的关联激活内-S检查点。 的EdU（+）的BrdU（ - ）核 埃杜（+）的BrdU（ - ）原子核对应于细胞，是在S期之前，而不是照射后。这些晶核Chromocenters为EdU（+），表明细胞为晚期S相当的EdU对小鼠注射（ 图3A）。由于NSPC的G2 / M期最后2小时在发育中的小鼠大脑中，埃杜（+）的BrdU（ - ）NSPC是在G2在0小时的PI。始终如一，最埃杜（+）的BrdU（ - ）原子核被发现在靠近心室在1小时PI的第一箱中未经辐照的大脑。这是NSPC的INM的结果，S期，其中许多人形成典型的有丝分裂象（ 图2B）之后。在1小时PI，埃杜数（+）的BrdU（ - ）细胞核被显著在心室表面降低，在照射大脑无丝分裂的数字是可检测的。这些数据清楚地表明细胞照射在G2中在1小时内的PI激活的G2 / M期检查点。此外，大多数的EdU的（+）的BrdU（ - ）这些晶核是凋亡，在4小时的PI表明NSPC是高度辐射敏感的G2（ 图2C）中。 的EdU（ - ）的BrdU（ - ）核 细胞与埃杜（ - ）的BrdU（ - ）原子核对应于两种细胞G0（主要是未成熟的神经元），或NSPC是留在G1从注册成立的EdU动物牺牲（ 图3E）。 1小时和4小时图2C的对比可以看出interkinetic核的迁移使这一说是接近脑室G1在1小时的细胞核进行他们的心尖到4小时基底迁移，而这是在S期细胞核去向着性室ular表面。 如图2C许多这些晶核是细胞凋亡，在4小时的PI。凋亡的EdU的数量（ - ）的BrdU（ - ）细胞核是最大的附近的心室表面及减少的上部箱。这些数据表明，因而该辐射诱导的细胞凋亡的发生NSPC期间早G1期和/或有丝分裂后的神经元迁移已进行照射。相比之下，没有或很少有凋亡细胞中发现了皮质板。这是根据已到达其最终目的地的神经元的辐射电阻。 埃杜（ - ）的BrdU（+）核 的EdU（ - ），BrdU阳性细胞核对应于输入的S期照射后的细胞。通常在1小时的PI，他们表现出常染色质弥漫性BrdU标记，但chromocenters不是。因此，他们在早S期当时（ 图3D）。在1小时的PI在S中马齿苋的数量和本地化的EdU电子箱（ - ）的BrdU（+）核是控制和辐照大脑相似。因此， 第一小时PI在S期进入并不受辐射小鼠发育中的大脑（ 图2B）。有趣的是，1小时和4小时的PI在照射大脑的比较表明中的EdU的数量下降（ - ），BrdU阳性细胞核和大部分这些晶核是细胞凋亡，在4小时的PI。这表明，该进入S期辐射后的细胞活化的帧内-S检查点和发生凋亡此后（ 图2C）。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51209/51209fig1highres.jpg" width="500" /> 图1:E14.5小鼠胚胎的大脑半球用DAPI染色。的EdU和BrdU染色以及核形态都在背内侧大脑的一个标准行业划分的部分脑冠状切面分析升壁使用分为18个垃圾箱（或更多）的网格，如文中所述。比例尺:20微米<a href="https://www.jove.com/files/ftp_upload/51209/51209fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51209/51209fig2highres.jpg" src="/files/ftp_upload/51209/51209fig2.jpg" /> 的照射的神经干细胞和祖细胞的图2。细胞周期进程（图从罗克等人 ，干细胞，2012）。实验设计和DAPI（蓝色）（一）示意图，埃杜（绿色），和BrdU（红色）发现冠状皮质切片在1和4小时的PI（2戈瑞）染色模式。 。比例尺= 20微米（B，C）埃杜数/ bin中（+）的BrdU（ - ），埃杜（+）的BrdU（+），埃杜（ - ）的BrdU（+），或埃杜（ - ）的BrdU（ - ）细胞核与正常莫rphology在未照射对照（蓝色），并与正常的（红色）或细胞凋亡（固缩，黑色）中照射的形态（2戈瑞）皮层在1小时（B）或4小时的PI（C）。在1小时的PI（二）未检测到凋亡细胞。使用邦费罗尼事后检验进行统计分析。缩写:的BrdU，5 - 溴-2&#39;-脱氧尿苷; DAPI，4&#39;-6-二脒基-2 - 苯基吲哚; EDU，5 - 乙炔基-2&#39;脱氧尿苷; IZ，中间地带; SVZ，脑室下区; VZ，脑室区。 <a href="https://www.jove.com/files/ftp_upload/51209/51209fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" fo:content-width="6in" fo:src="/files/ftp_upload/51209/51209fig3highres.jpg" src="/files/ftp_upload/51209/51209fig3.jpg" /> 图3差分的EdU和常染色质和chromocenters上面板的BrdU染色:Z堆栈埃杜（绿色）的共聚焦图像，的BrdU（红）染色和DNA经DAPI染色（蓝色），让chromocenters为明亮的蓝色灶的检测。染色是对在那里的EdU被交付1.5小时实验1小时PI收集胚胎大脑部进行照射和BrdU 0小时前的PI（只未照射控制显示）（比例尺:5微米）。 <a href="https://www.jove.com/files/ftp_upload/51209/51209fig3highres.jpg" target="_blank">请点击这里查看更大的版本这个数字。</a> 底部面板显示的EdU和冠状脑切片检测到的BrdU染色的5种不同模式的例子。合并图像和单通道显示（比例尺:2微米）: A）核与埃杜（+）chromocenters和埃杜（+）或埃杜（ - ）常染色质。的EdU一直从而在后期S期臂间异染色质的复制过程中掺入:细胞是我Ñ​​G2在0小时的PI。 B）核与埃杜（+）常染色质和BrdU（+）chomocenters:的EdU已经在S早期阶段被纳入当大多数常染色质的复制和BrdU已晚S期被纳入:将细胞在S期在0小时PI 。 C）核与埃杜（+）的BrdU（+）常染色质和埃杜（ - ）的BrdU（ - ）chromocenters。的EdU和BrdU已被纳入S期的第一部分:将细胞在S早期阶段在0小时的PI。 D）核用BrdU（+），常染色质和埃杜（ - ）的BrdU（ - ）染色中心。的BrdU已经在S阶段的开始被纳入。该细胞在G1后期在0小时PI和进入S期之后。 E）核与埃杜（ - ）的BrdU（ - ）常染色质和chromocenters。该细胞在G0（未成熟的神经元），或在G1期，期间的EdU和BrdU脉冲没有进入S期。 <table b为了="0"cellpadding的="0"CELLSPACING ="0"宽度="415"&gt; <tbody><tr><td></td><td>解</td><td>孵化</td><td>温度</td><td>压力</td><td>搅动</td></tr><tr><td> 1 </td><td>醇70％ </td><td> 30分钟</td><td> 35°C </td><td>上</td><td>上</td></tr><tr><td> 2 </td><td>酒精95％ </td><td> 15分钟</td><td> 35°C </td><td>上</td><td>上</td></tr><tr><td> 3 </td><td>酒精95％ </td><td> 30分钟</td><td> 35°C </td><td>上</td><td>上</td></tr><tr><td> 4 </td><td>酒精95％ </td><td> 45分钟</td><td> 35°C </td><td>上</td><td>上</td></tr><tr><td> 5 </td><td>酒精100％ </td><td> 15分钟</td><td> 35°C </td><td>上</td><td>上</td></tr><tr><td> 6 </td><td>酒精100％ </td><td> 30分钟</td><td> 35°C </td><td>上</td><td>上</td></tr><tr><td> 7 </td><td>酒精100％ </td><td> 1小时</td><td> 35°C </td><td>上</td><td>上</td></tr><tr><td> 8 </td><td>甲苯100％ </td><td> 30分钟</td><td> 35°C </td><td>上</td><td>上</td></tr><tr><td> 9 </td><td>甲苯100％ </td><td> 45分钟</td><td> 35°C </td><td>上</td><td>上</td></tr><tr><td> 10 </td><td>甲苯100％ </td><td> 1小时</td><td> 35°C </td><td>上</td><td>上</td></tr><tr><td> 11 </td><td>石蜡</td><td> 30分钟</td><td> 58°C </td><td>上</td><td>上</td></tr><tr><td> 12 </td><td>石蜡</td><td> 1小时</td><td> 58°C </td><td>上</td><td>上</td></tr><tr><td> 13 </td><td>石蜡</td><td> 1小时</td><td> 58°C </td><td>上</td><td>上</td></tr><tr><td> 14 </td><td>石蜡</td><td> 1.5小时</td><td> 58°C </td><td>上</td><td>上</td></tr></tbody></table> 表石蜡包埋1。真空渗透处理程序。 <table border="0" cellpadding="0" cellspacing="0" width="311"><tbody><tr><td>反应组分1幻灯片</td><td></td></tr><tr><td> 1X的EdU反应缓冲液</td><td> 128.64微升</td></tr><tr><td> 硫酸铜 </td><td> 6微升</td></tr><tr><td>的EdU的Alexa Fluor叠氮化物</td><td> 0.36微升</td></tr><tr><td> 1X的EdU反应缓冲添加剂</td><td> 15微升</td></tr><tr><td> 总成交量 </td><td> 150微升 </td></tr></tbody></table> 表2。的EdU反应鸡尾酒。

Watch this Scientific Journal Video about Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress at JoVE.com

评估基因毒性应激后小鼠大脑发育中神经干细胞和祖细胞的细胞周期进展神经科学 |视频

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

胸苷两个类似物管理，埃杜和BrdU，在孕鼠允许在神经细胞和祖细胞在胚胎小鼠大脑细胞周期进程的分析。这个方法是有用的，以确定基因毒性应激，包括电离辐射，脑发育过程中的作用。

神经干细胞和祖细胞在小鼠脑开发基因毒性应激后的评估细胞周期进程

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

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Nanyang Technological University

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Cell Cycle and Division

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15.2K 次观看.  CEA DSV iRCM SCSR. 胸苷两个类似物管理，埃杜和BrdU，在孕鼠允许在神经细胞和祖细胞在胚胎小鼠大脑细胞周期进程的分析。这个方法是有用的，以确定基因毒性应激，包括电离辐射，脑发育过程中的作用。 

视频: Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

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

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer treatments as used in clinical oncology. It enables a quantitative and longitudinal analysis of the DNA damage response after genotoxic stress, as induced by radiotherapy and a multitude of anti-cancer drugs. The method covers all stages of the DNA damage response, providing endpoints for induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and cell death by apoptosis in case of repair failure. Combining these measurements provides information about cell cycle-dependent treatment effects and thus allows an in-depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. As the effect of many cancer therapeutics including chemotherapeutic agents and ionizing radiation is limited to or strongly varies according to specific cell cycle phases, correlative analyses rely on a robust and feasible method to assess the treatment effects on the DNA in a cell cycle-specific manner. This is not possible with single-endpoint assays and an important advantage of the presented method. The method is not restricted to any particular cell line and has been thoroughly tested in a multitude of tumor and normal tissue cell lines. It can be widely applied as a comprehensive genotoxicity assay in many fields of oncology besides radio-oncology, including environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells.

The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.

细胞周期特异性测量βH2AX和细胞凋亡后热源毒性应力通过流细胞测定

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

癌症研究

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

将人诱导的多能干细胞分化为神经元和神经胶质的混合培养物进行神经毒性测试的方案

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

人类神经的聚集体cGMP的适用展开法干细胞和祖细胞来源从多能干细胞或胎儿脑组织

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

神经科学

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

作为一种工具来描绘分子途径参与神经毒性和神经保护神经元细胞在线实时阻抗为基础的细胞分析仪

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

建模星形细胞瘤发病机制<em&gt;在体外</em&gt;和<em&gt;体内</em&gt;使用星形胶质细胞或条件，遗传工程小鼠神经干细胞

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

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

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

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

神经干细胞和祖细胞来自成年小鼠脑室下区细胞分选和实时成像的细胞周期动力学

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

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 ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

小鼠脊髓损伤模型中内源性神经干细胞活化的干细胞检测

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

成年小鼠大脑中诱导荧光标记干细胞的谱系示踪

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...