Name: temporal tracking of cell cycle progression using flow cytometry without the need for synchronization
Uploaded: 2015-08-16T13:00:00.000+00:00
Duration: 8 min 52 s
Description: Watch this Scientific Journal Video about Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization at JoVE.com

The work was funded by the Leukemia and Lymphoma Society of the USA (6105-08), a Cancer Council NSW grant (13-02), an NHMRC Senior Research Fellowship (LJB) (1042305) and project grant (1041614).

这里所描述的协议使用的急性淋巴细胞白血病细胞系NALM6但可以适用于其他类型的细胞。 1.解决方案和试剂<ol><li>完全RPMI <ol><li>添加56毫升胎牛血清（FCS）和毫升的200mM 5.5 L-谷氨酰胺至500毫升瓶的RPMI-1640培养基。 </li></ol></li><li> BrdU的库存解决方案<ol><li>在Dulbecco氏磷酸盐缓冲盐水（DPBS）制备32.5毫BrdU的（10毫克/毫升）。 </li></ol></li><li>尿嘧啶完全RPMI <ol><li>加入6.2微升的BrdU原液中加入10毫升完全RPMI中。 </li></ol></li><li> DNA酶解决方案<ol><li>制备1毫克DNA酶/ ml，在DPBS。 </li></ol></li><li>染色缓冲液<ol><li>制备3％热灭活的FCS和0.09％叠氮化钠在DPBS中。 </li></ol></li><li>请参阅材料清单固定缓冲液，透化缓冲液和洗涤液的定义。 </li></ol> 2.细胞没有德:将细胞未培养大于6个月。这种方法是直接适用于与调整细胞密度和培养介质中的任何非粘附细胞系。使用的增长指数在实验开始细胞。 <ol><li>保持NALM6细胞在完全RPMI T-75培养瓶中。执行使用Ⅱ级生物安全柜无菌条件下的所有步骤。 <ol><li>保持NALM6细胞每毫升之间1-2×10 6细胞每周三次分裂的文化。 </li><li>在5％的孵育在37℃ 的 CO 2在空气中。 </li></ol></li></ol>细胞的BrdU 3.脉冲标记小心:处理BrdU的小心，因为它是一个潜在的致突变和致畸。 <ol><li>离心细胞，在150×g离心5分钟。注意:传输细胞进入新鲜培养基提高了结果的重现性。 </li><li>以2×10 6 C进行细胞计数和重悬的细胞在完全的RPMIELLS /毫升。 </li><li>稀释细胞1 2用BrdU完全RPMI产生最终细胞的1×10 6个细胞/ ml的浓度。 </li><li>在37℃，5％CO 2的45分钟，然后稀释细胞1在10与完全RPMI。离心细胞，在150×g离心5分钟，并小心地丢弃所有上清液。 </li><li>重悬的细胞在完全的RPMI小体积（〜100微升），进行细胞计数，并调整到1×10 6个细胞/ ml。 </li><li>吸管1毫升细胞向一个48孔平板的孔中。吸取1毫升DPBS到任何无人居住的井获得更多的可重复的结果。 </li><li>在5％CO 2在37℃的空气中对希望的时间点，在这里1，2，3，4，5，6，7，8，9，10，11，12，13，14，15，16，17 ，18，19，20，21，22，23和24小时。注意:的时间长度将取决于实验设计的目的在于测量。 </li><li>传送所有的细胞变成使用移液管FACS管。用1ml VOLU冲洗顺序以及的PBS MES至5 ml的最终总体积。 </li><li>离心150 XG 5分钟，然后小心地取出所有的上清。细胞准备进行染色，执行该立即（第4节）。 </li></ol> 4.细胞染色注:如果需要的细胞表面染色固定之前执行它，以确保将细胞保持在4℃下贯穿。 <ol><li>重悬的细胞在100μl的染色缓冲液（用于可选表面染色，加入抗体的推荐量对表面抗原和温育30分钟，在4℃）。 </li><li>加入1毫升的染色缓冲液中，离心5分钟，在150×g离心并弃去上清液。 注意:特定抗体，浓度，培养时间等，将取决于具体的实验目标而有所不同。 </li><li>固定和透<ol><li>重悬的细胞在100μl的固定缓冲液孵育15分钟，在室温下进行。 </li><li>加入1毫升邻f。洗涤缓冲液，离心5分钟，在150×g离心并弃去上清液。 </li><li>重悬的细胞在100μl的透化缓冲液孵育的细胞在冰上10分钟。 </li><li>加入1毫升的洗涤缓冲液，离心5分钟，在150×g离心，并弃去上清液。 </li><li>重悬的细胞在100微升每管固定缓冲孵育5分钟，在室温下进行。 </li><li>加入1毫升的洗涤缓冲液，离心5分钟，在150×g离心，并弃去上清液。 注意:如果需要，该协议可以在这里暂停。固定细胞可稳定数天，在4℃，如果再悬浮于染色缓冲液中。取出染色缓冲以下，然后再继续离心。 </li></ol></li><li> DNA酶处理<ol><li>重悬的细胞在100μl的DNA酶溶液（30微克DNA酶/ 10 6个细胞），并孵育细胞1小时，在37℃。 </li><li>加入1毫升的洗涤缓冲液，离心机在150×g离心5分钟并弃上清。 </LI&gt; </ol></li><li>抗体染色 注意:染色比的BrdU其它胞内标记物可同时与BrdU的染色来进行。 <ol><li>重要提示:准备补偿控制，包括未染色的细胞和细胞标记每个单独的荧光染料。理想情况下，使用相同的抗体用于补偿控制那些在实验管中使用。但是，如果这是不可行的，替代的抗体高度表达的抗原缀合到相同的荧光染料。 </li><li>将细胞重悬于50微升的洗涤缓冲液，并添加1微升/ 10 6个细胞的BrdU抗体。注意:直接缀合抗体对其他特定细胞内抗原也可加入。 注:抗体对组蛋白H3磷酸化上Ser10可用于细胞在G2和M之间进行区分，组蛋白H3磷酸化在Ser10有丝分裂过程10的抗体的cdc2磷酸上Tyr15可用于检测细胞甲肝Ë致力于有丝分裂。11 </li><li>孵育细胞20分钟，在室温下进行。 </li><li>加入1毫升的洗涤缓冲液，离心细胞，在150×g离心5分钟并弃去上清。 </li></ol></li></ol><ol start="6"><li>对于细胞周期分析染色的DNA <ol><li>松开颗粒和添加7-AAD溶液（0.25微克）的20微升。注意:关键是要使用的7-AAD /细胞的恒定量。 </li><li>重悬在1ml染色缓冲液中的细胞。 </li></ol></li></ol> 5.收集流式细胞仪数据注意:需要将依赖于所使用的荧光染料的数量和性质的机器。 <ol><li>收集以下参数:FSC-A，SSC-A，FSC-H（FSC-W能够被用来代替FSC-H）和7-AAD荧光上的线性标尺。收集对数尺度的APC渠道。收集所需的使用对数刻度的表面或内部的标签评估任何额外的通道。 </li><li>执行COM补偿重叠在分析样品之前不同的荧光染料之间观察到的发射光谱信号。注意:大多数的流式细胞仪将自动执行此。 </li><li>收集至少10000个事件用于每个样品。 </li></ol>流式细胞仪数据分析6. 注意:的FlowJo用于本研究用于流式细胞术数据的分析，但其他软件包也可以使用。门控策略示于图1。 <ol><li>使用FSC-A和SSC-A参数标识的活细胞群。 </li><li>在这一群体中排除双峰和使用的FSC-A和FSC-H（FSC-W这里也可以使用）的聚集体。 </li><li>在此人口设置使用7-AAD对x轴和BrdU-APC在y轴的点图。 </li></ol><img alt="图1" src="/files/ftp_upload/52840/52840fig1highres.jpg" width="700" /> 图1:门控策略左PA。道:非门的细胞都显示在一个FCS-A对SSC-A点图。活细胞群体确定所示的门。中心板:细胞从左侧面板选通显示在一个FSC-A对FSC-H散点图（FSC-W能够被用来代替高度）。双峰和聚集体识别，排除所示的门。右面板:细胞在中心面板上的双重排斥日期控显示在7-AAD与APC-A点图。所述的BrdU抗体用的APC允许细胞已掺入的BrdU脉冲标记期间的标识。 7-AAD提供对DNA含量的信息。上闸门定义S期细胞阳性尿嘧啶，因此在BrdU的脉冲，左下门，细胞在G中0/1与下右闸的G中时2 / M。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52840/52840fig1highres.jpg" target="_blank">请点击此处查看更大的版本这个数字。</a> <ol start="4"><li>的细胞周期<html> nalysis <ol><li>打开对细胞中的双峰排除门的第一数据文件和栅极。 </li><li>分析这一群体的细胞周期分布（位于下FloJo软件平台），并使用迪安 - 杰特 - 福克斯模型。 </li><li>获得对G 0/1和G使用create门2 / M峰的位置。 </li><li>门上的BrdU阳性细胞及受这个人口同一个细胞周期分析。 </li><li>通过从创建栅极施加相同的栅极和设置约束（用形成的门），在G 0/1和G 2 / M期峰的位置提供对G 0/1和G 2 / M期峰的位置。这表示在所述第一2板图2。 注意:其它软件也可用于分析数据和指令将相应地变化。 </li></ol></li></ol>840 / 52840fig2highres.jpg"WIDTH ="700"/&gt; 图2:细胞周期进程的第一面板（全细胞）被选通由所述双峰排除门所限定的细胞群。这一人群用7-AAD对X轴显示以直方图。对G 0/1峰的峰值是由轴下方的箭头表示。在后续板BrdU阳性细胞已被选通打开， 如图1。为双峰排除栅极选通时获得对G 0/1位置的值被施加到内的FlowJo细胞周期软件的BrdU阳性门控细胞。每个随后的面板被选通的BrdU阳性人口如图1和基于当分析整个人群中所示的前两个板得到的值对G 0/1峰的位置。使用的BrdU阴性部分以识别为BrdU阳性细胞在相同的SAMP对G 0/1人口的位置勒控制用于在样品间DNA的染色强度的任何微小的差别。每个面板上显示的数字表示自从BrdU的脉冲结束的时间。计算的细胞周期显示在绿色阴影。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52840/52840fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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The authors have nothing to disclose.

分析细胞周期的能力，对于癌症生物学的理解和两种药物和基因影响细胞增殖和生长的作用机制重要。虽然有许多测定法，据说测量细胞增殖的，多数只提供的量度指示的数目的细胞存在。这些包括测量通过直接可视化和计数，代谢活性或ATP浓度细胞数测定。许多这些方法的主要优点是，它们相对容易地进行，并适合于微孔板格式和自动化，使它们可用于筛选大量的条件或化合物。许多这些方法的缺点是，由于死亡的细胞损失不考虑，可能导致细胞增殖的低估。此外，这些方法测量的体积人口和不允许单个细胞的研究或细胞通过中转细胞周期。 常用的增殖测定，也许是最可靠和准确的是那些测量DNA的合成。传统的细胞增殖测定孵育细胞为几个小时至过夜用3 H-胸苷，其被掺入新合成的DNA。13这一方法的明显的问题是利用放射性材料，但另一个限制是，该结果测量平均细胞群的增殖。的BrdU同样可以使用未经辐射的问题，虽然还需要一些步骤和BrdU是一个潜在的致变物。然而，BrdU的具有可与流兼容术，允许单个细胞的分析的优点。14其他流式细胞兼容的方法来评估细胞增殖，在单细胞水平包括越来越多的标记的细胞膜或细胞蛋白质染料（ 如 CFSE）daught之间产生分歧呃细胞，DNA嵌入染料和细胞周期特异性抗原检测由抗体。的细胞标记染料的最佳允许细胞分裂的跟踪过一些细胞分裂。15它们提供了一个直接衡量扩散，虽然没有获得关于细胞周期阶段或分布信息。 DNA含量，使用DNA嵌入染料如碘化丙啶或7-AAD提供强的细胞周期分布16，但不是时间的，数据的测量。即使有多个时间点评估它仍然不可能评估细胞周期或特定的细胞周期阶段的长度。细胞周期特异性抗原可用于评估在非同步的细胞群的细胞周期阶段。常用细胞周期特异性抗原包括Ki-67的，17是在S期间表达，G 2和细胞周期的M期但不是G 0/1，PCNA（增殖细胞核抗原）18，和磷酸化小时内istone H3。19虽然这些提供关于细胞周期阶段与细胞周期的动态缺乏良好的数据，信息。 对细胞周期动力学获得数据在传统上被审查通过同步用试剂或阻断细胞周期进展的培养条件的细胞。这将导致块，一旦去除，导致波的细胞通过细胞周期进展一起的背后细胞的积累。1细胞周期的持续时间和各种细胞周期的长度的计算是然后可能的。细胞可以被诱导退出活跃的细胞周期进入摹0通过血清剥夺。在重新添加血清的细胞然后可以一起移动到后续阶段。20虽然这对于某些类型的细胞，其他人，包括许多转化细胞类型的可靠方法，不能退出细胞周期和频繁经历细胞死亡作为结果21事实上，我们STU模具发现这是急性淋巴细胞白血病细胞的情况。此外，细胞通常不能重新进入细胞周期的足够同步的方式。化学方法可用于诱导细胞周期停滞在细胞周期的特定阶段。例如，活性剂，以防止DNA合成（ 如过量胸苷）或阻止有丝分裂纺锤体的形成（ 如诺考达唑）阻滞细胞的S和M期分别，但是有毒的，可能会导致生长紊乱，甚至死亡的一个显著比例细胞22在ALL细胞这些方法未能阻止多数细胞而不杀死一个显著分数。考虑到目的是评估对细胞周期参数的潜在的抗白血病剂的效果，从同步造成的细胞死亡是不可接受的。尽管有大量的方法的描述来同步细胞，都有缺点。存在的主要问题是:不足富集的细胞在细胞周期的所需部分，干扰细胞周期的正常生理或，如观察到的，过量的毒性。 这里所描述的方法是长期使用脉冲系统，其中细胞温育一个短暂的时期用BrdU查明S期细胞的简单扩展。我们发现了一个45分钟的脉冲是在我们的系统中最佳但如果获得足够的标记更短的时间周期可以被使用。通过去除的BrdU后继续培养细胞，可以通过在S期，G 2，有丝分裂中，G 1和条目的剩余部分进入下一细胞周期跟踪他们的进展。有可能以进一步跟随细胞。这个系统的优点是，没有证据毒性的细胞中这些实验的时间帧的并且存在着对细胞的生长干扰最小，因为只有一对夫妇的媒体的变化是必需的。将细胞在孔蒂否则保持nuous文化。术的方法的基础的性质，流动装置，它可与细胞亚群由附加的表面或细胞内染色的识别进行组合。我们使用的APC缀合的BrdU抗体，但其它缀合物可以被取代，以促进抗体的面板的开发。我们用7-AAD，而不是碘化因为7-AAD在荧光单通道最大化的多色染色方案。同样DAPI或Hoechst的可以用作DNA渍，但需要一个紫外激光器。对于DNA染色更紧简历可以用乙醇固定获得但这经常损害抗体染色，限制了额外的表面或细胞质污渍的选项。最大的缺点是S期持续约8小时，这样标记的细胞可能刚刚进入或即将退出在脉冲周期S期。其结果是，该同步是不紧张，因为与一些其它系统。然而，通过BrdU标记与染料指示的DNA结合内容和抗体特异性细胞周期依赖性蛋白，强数据可以得到。 为了获得一致的数据，以确保该细胞是在正确浓度和跨越被比较的所有条件的细胞浓度是一致是重要的。审慎的7-AAD染色的亮度，以细胞浓度非常敏感。使用自动细胞计数系统可以帮助确保细胞浓度是所有样品在过程中的每个步骤中保持一致。另外重要的一点是时间的细胞暴露于BrdU的长度。小的变化可以转化为相当大的差异，因此，重要的是，所有样品孵育用BrdU为正好在同一时间。最后，为了滴定抗体，以获得最佳的染色是很重要的，这应被重复每当批次变化。如果遵循所有的步骤和一贯的细胞浓度保持不变，那么这种实现方法具ð将可靠地使细胞通过细胞周期的跟踪而无需同步。也有相当大的空间来修改这个方法以适应特定的细胞类型和解决具体问题。

的细胞周期功能和发生在细胞的变化过程中的细胞周期进展的评估是基本理解生物学，特别是癌症生物学的许多方面。在发展为恶性肿瘤的治疗许多药物对细胞周期进程产生深远的影响，或通过细胞周期依赖性机制诱导细胞死亡。为了研究细胞周期动力学或细胞在细胞周期的特定阶段中，通常以同步的细胞。然而同步方法可以对细胞有害作用被研究，有可能混淆的结果。1在单细胞随时间的细胞周期进程的最新的使用荧光标记的蛋白质，是只存在于细胞周期的特定阶段的已批准的分析2，但是所述细胞进行研究需要被遗传操纵以表达这些标记的蛋白质，限制了其使用的地方这可以读取系统容易完成。 细胞周期包括两个活性阶段:合成（S）相，其中，DNA复制和有丝分裂（M），其中，细胞分裂发生。这些阶段由三个间隙相，G 0，G 1和G 2分开。 ģ0或静止，是其中细胞已离开循环的休止阶段中，G 1是其中细胞大小之前的DNA复制和G 2，其中的DNA复制的完成之间但细胞分裂前的细胞生长继续增加。通过细胞周期的进展是由多个检查站的控制。当环境条件不支持DNA合成和防止进入S期对G 1检查点被激活。内-S期检查点或延迟可以通过DNA损伤可能会导致复制叉停滞了被触发。期间克2-复制的DNA的保真度被确认，并且如果检测到损坏那G 2 </子&gt;检查点被激活允许细胞分裂DNA修复之前。有丝分裂期间的最后的检查点可确保染色单体已经在有丝分裂板被正确地对准，使得细胞分裂可顺利完成。这些检查点的3激活通常用于同步的细胞群。细胞周期关卡可以通过许多因素，但在肿瘤生物学中最常见的是检测DNA损伤激活。 DNA损伤响应由PI3激酶样激酶共济失调毛细血管扩张症和RAD3相关（ATR）发起和共济失调毛细血管扩张症突变（ATM），该激活下游效应激酶Chk1的和Chk2，分别3事件的范围激活的Chk1包括停滞复制叉，DNA的交联，和紫外线辐射的伤害而Chk2的是由双链断裂主要激活。 为研究对细胞周期i的长度的改变的条件下的效果的常用方法s到同步细胞在细胞周期的特定阶段。1这可以通过几种方法来实现。细胞可被物理分离基于尺寸，密度，侧向散射（粒度），以及细胞表面表达的标志。更实际，细胞可以通过化学手段进行同步。几剂，例如胸苷，羟基脲和阿糖胞苷可用于抑制DNA合成的细胞周期导致S期细胞继续循环的剂被除去后的累积的S期。细胞治疗诺考达唑，这防止形成有丝分裂纺锤体，逮捕与G 2 -或M相DNA含量。消除血清从培养基导致细胞于G1 0相位的累积。再加入内培养血清的营养素重新启动细胞的正常循环。然而，所有的这些同步方法干扰细胞的正常循环和生长，并且可以resul吨显著细胞死亡。 急性淋巴细胞白血病细胞的同步是特别具有挑战性的，并且这些细胞不适合于遗传操作。这里所描述的方法允许细胞周期动力学的评估和细胞在细胞周期的特定阶段没有传统同步或遗传修饰的研究。这种方法还可以用于其中，遗传修饰和传统的同步过程不容易实现其他类型的细胞是有用的。该方法是基于长期建立的使用溴脱氧尿苷（尿嘧啶）掺入，这对短期细胞的生长和增殖的影响非常小的。4成立的BrdU协议利用的BrdU的掺入在S期新合成的DNA 。这标志着永久细胞的BrdU曝光时已经处于S期。这部分人群可在稍后的时间点被染色的BrdU incorpor确定通货膨胀和由此充当可以遵循并评估在时间允许的对细胞周期转运药物作用的研究同步人口。 BrdU的需要前 ​​抗体染色要曝光，通常是达到以下DNA酶或酸处理。6,7-使用流式细胞仪来检测掺入的BrdU使包括额外的标记。最重要的是利用染料来测量DNA含量，使该人在S期在研究开始时的细胞的细胞周期时相分布的评估。8另外附加的表面或细胞内的抗原，也可以研究。9，这些可能与细胞周期事件，如Ki67的或看似无关的细胞功能，如细胞凋亡的标记一样裂解的caspase-3。潜在的应用是由研究者的想象力的限制。

<table><tbody><tr><td>APC BrdU Flow Kit</td><td>BD Biosciences</td><td>552598</td><td>Contains BrdU antibody, 7-AAD and BD Cytofix/Cytoperm&lt;br/&gt; Buffer (referred to as Fixation Buffer)</td></tr><tr><td>BD Cytoperm Permeabilization Buffer Plus</td><td>BD Biosciences</td><td>561651</td><td>Referred to as Permeabilization buffer</td></tr><tr><td>BD Perm/Wash Buffer</td><td>BD Biosciences</td><td>554723</td><td>Referred to as Wash buffer</td></tr><tr><td>DNase</td><td>Sigma</td><td>D-4513</td><td></td></tr><tr><td>BD Falcon 12 x 75&amp;nbsp;mm FACS tubes</td><td>BD Biosciences</td><td>352008</td><td></td></tr><tr><td>BD Pharmingen Stain Buffer</td><td>BD Biosciences</td><td>554656</td><td></td></tr><tr><td>BD LSR FORTESSA flow cytometer</td><td>BD Biosciences</td><td>FORTESSA</td><td></td></tr><tr><td>Pipetman</td><td>Gilson</td><td>P2, P20, P100, P1000</td><td></td></tr><tr><td>RPMI 1,640 w/o L-Gln 500 ml</td><td>Lonza</td><td>12-167F</td><td></td></tr><tr><td>DPBS</td><td>Lonza</td><td>17-512F</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>FisherBiotec</td><td>FBS-7100113</td><td></td></tr><tr><td>L-Glutamine</td><td>Sigma</td><td>G7513-100ML</td><td></td></tr><tr><td>5-Bromo-2&amp;prime;-deoxyuridine</td><td>Sigma</td><td>B5002-1G</td><td></td></tr><tr><td>Falcon TC 150&amp;nbsp;cm&lt;sup&gt;2&lt;/sup&gt; vented Flasks</td><td>BD Biosciences</td><td>355001</td><td></td></tr><tr><td>Pipettes 25&amp;nbsp;ml</td><td>Greiner</td><td>760180</td><td></td></tr><tr><td>Aersol Pipettes 200&amp;nbsp;&amp;micro;l</td><td>Interpath</td><td>24700</td><td></td></tr><tr><td>Aersol Pipettes 1&amp;nbsp;ml</td><td>Interpath</td><td>24800</td><td></td></tr><tr><td>Centrifuge</td><td>Spintron</td><td>GT-175R</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Binder</td><td>C 150</td><td></td></tr><tr><td>AF488 anti-Histone H3 Phospho (Ser10) Antibody</td><td>Cell Signalling</td><td>9708S</td><td></td></tr><tr><td>Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb</td><td>Cell Signalling</td><td>2197S</td><td></td></tr><tr><td>Phospho-Chk1 (Ser345) (133D3) Rabbit mAb</td><td>Cell Signalling</td><td>2348S</td><td></td></tr><tr><td>NALM6&amp;nbsp;</td><td>DSMZ</td><td>ACC-128</td><td></td></tr></tbody></table>

temporal tracking of cell cycle progression using flow cytometry without the need for synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

这个方法可以用于获得信息的范围。一些应用程序在这里列出。 细胞周期的持续时间的评估为了确定通过细胞周期所需的细胞过境时间，将细胞收获在以下的BrdU的脉冲不同的时间点。评估之间的时间间隔，可以适应于特定的细胞被分析。造血细胞系进行了评估每小时超过在不存在任何药物治疗，以确定细胞周期阶段标准培养条件下的长度的24小时期间， 图2示出了一个选择NALM6细胞在24小时内的快照的分析。将细胞在所述的BrdU脉冲（ 即内图1中的BrdU阳性栅极）时S期进展至G 2 / M期，随着细胞在细胞周期被的相的峰值检测10小时的BrdU的p后ulse。脉冲后，S期的BrdU标记细胞的比例达到了最低点14小时，几乎所有的细胞都17小时后返回至G 1。通过24小时的BrdU阳性细胞的比例已重新进入S期作为其下一个细胞周期的一部分，指示细胞可以内完成24小时的细胞周期，但是大多数还没有进入第二轮的复制，与已知的36一致小时的倍增时间为这些细胞。 细胞周期分布可以通过包含附加的标记物来进一步细化。例如，加入一种抗体组蛋白H3磷酸化上Ser10（细胞中有丝分裂的标记物）的允许细胞在从那些在G中2有丝分裂的判别（左图3的面板）。 <img alt="图3" src="/files/ftp_upload/52840/52840fig3highres.jpg" width="700" /> 图3:确认细胞周期阶段使用其他标记，上图板显示从BrdU阳性栅极细胞的细胞周期分析， 如图1，细胞已被孵育的为3nM长春新碱的存在或不存在的BrdU的脉冲之后18小时。 如图2所示的细胞周期分析进行的。较低的面板显示与组蛋白H3的7-AAD同一细胞上的磷酸化点Ser10情节。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52840/52840fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 药物作用对细胞周期评估细胞毒性剂可以具有对细胞周期进程产生深远的影响，并且可以诱导细胞死亡。药物对细胞周期进程的效果可通过以下所述的BrdU脉冲附加感兴趣的药物进行评估。示出两个例子。首先是一种情况，感应Ø˚F细胞死亡混淆的细胞周期的数据进行分析（ 图3的右图）。 NALM6细胞已经暴露为3nM长春新碱18小时BrdU的脉冲之后。细胞有望在有丝分裂逮捕长春新碱目标微管。然而，细胞周期分析表明，这些细胞还没有被捕，但通过与G 0/1（ 图3右上图）过渡。增加一个抗体组蛋白H3磷酸化在Ser10的显示，细胞没有退出有丝分裂（ 图3右下图），尽管有减少的DNA含量。它很可能是该DNA含量下降，因为细胞经历凋亡，并开始降解它们的DNA。由于细胞启动细胞凋亡，而在有丝分裂（ 即用4N DNA），它们显示为S期或G 0/1细胞而不是典型的凋亡分G 1个峰。 <img alt="图4" src="/files/ftp_upload/52840/52840fig4highres.jpg" width="700" /> 图4:检测细胞周期的特定阶段查杀散点图显示剩余的BrdU的脉冲后的培养24小时BrdU阳性细胞的百分比。细胞已被培养在存在或不存在车辆的单独（对照），1.5或16μMRAD001由于BrdU的脉冲结束。较低的面板显示细胞是BrdU阳性在24小时培养的百分比。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52840/52840fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 在另一实例中， 图4示出了如何在RAD001（mTOR抑制剂）细胞的低浓度的存在下能完成细胞周期，但然后行G中0/1的延迟或停滞。 RAD001较高浓度的主要是阻止细胞TRAnsiting通过与G 0/1。重要的细胞处于S期（ 即 BrdU阳性）被证明优先从培养时消失RAD001溶液。这表明，细胞通过G2 / M更容易受到此代理细胞死亡。12 另外，也可以检查细胞在使用抗体特异性抗原的细胞周期的特定阶段的反应。 图5中的细胞周期关卡调控蛋白的Chk1和Chk2响应于长春新碱治疗的激活被示出。 Chk1的和Chk2被磷酸化Ser345和Thr68分别激活。的Chk1和Chk2的活化可以看出，在出现具有2N DNA含量的细胞，但如图5所示很可能是细胞的有丝分裂即已经开始DNA降解为细胞凋亡的结果。 有可能在以后的不同时间点添加药物BrdU的脉冲检查试剂对细胞在细胞周期的各个阶段的影响。 <img alt="图5" src="/files/ftp_upload/52840/52840fig5highres.jpg" width="700" /> 图5:检测变化对在特定细胞周期阶段的细胞上图表示细胞在7-AAD对磷酸化的Chk1（左）或Chk2的（右）点图门控的BrdU阳性细胞如在图1中。以下所述的BrdU脉冲中的为3nM长春新碱的存在或不存在作为用于图3中的细胞已经过培养18小时。在较低的面板显示选通或者作为指示的2N或4N级分相同的细胞的重叠的直方图。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52840/52840fig5highres.jpg" target="_blank">请按此查看该图的放大版本。</a>

Watch this Scientific Journal Video about Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization at JoVE.com

使用流式细胞术对细胞周期进展进行时间跟踪，无需同步 |生物学 |视频

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

这个协议描述了使用溴脱氧尿苷（BrdU）标记的摄取，以允许细胞是在S期在时间上的特定点的时间的跟踪。此外DNA染料和抗体标记的方便了S期细胞的命运在稍后时间详细分析。

细胞周期进程的时间跟踪使用流式细胞仪，无需同步

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

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Nanyang Technological University

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Biology

Cellular Processes

Cell Cycle and Division

SCIENTISTS IN ACTION

19.1K 次观看.  Westmead Millennium Institute for Medical Research and University of Sydney. 这个协议描述了使用溴脱氧尿苷（BrdU）标记的摄取，以允许细胞是在S期在时间上的特定点的时间的跟踪。此外DNA染料和抗体标记的方便了S期细胞的命运在稍后时间详细分析。 

视频: Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

通过两个互补细胞同步方案研究细胞周期调控的基因表达

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

科研

遗传学

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

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

基于同步成像和流式细胞术检测治疗诱导的衰老癌细胞中的多种荧光衰老标志物

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

通过细胞周期激酶和磷酸酶活性成比例的FRET监测

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

生物学

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

代谢标记和流式细胞仪测定细胞周期进程的动力学

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able to generate large datasets, necessitate the destruction of the cell of interest, making it impossible to assess its functional fate. Heterogeneous biological differentiating systems like hematopoiesis are therefore difficult to describe. Building on cell division tracking dyes, we further developed a protocol to simultaneously determine kinship, division number, and differentiation status for many single hematopoietic progenitors. This protocol allows the assessment of the ex vivo differentiation potential of murine and human hematopoietic progenitors, isolated from various biological sources. Moreover, as it is based on flow cytometry and a limited number of reagents, it can quickly generate a large amount of data, at the single-cell level, in a relatively inexpensive manner. We also provide the analytical pipeline for single-cell analysis, combined with a robust statistical framework. As this protocol allows the linking of cell division and differentiation at the single-cell level, it can be used to quantitatively assess symmetric and asymmetric fate commitment, the balance between self-renewal and differentiation, and the number of divisions for a given commitment fate. Altogether, this protocol can be used in experimental designs aiming to unravel the biological differences between hematopoietic progenitors, from a single-cell perspective.

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Presented here is a flow cytometry-based technique that allows for simultaneously measuring the number of cell divisions, surface cell phenotype, and cellular kinship. Those properties can be tested statistically using a permutation-based framework.

通过流式细胞术 同时 评估造血干细胞和祖细胞的亲缘关系、分裂数和表型

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able ...

免疫与感染

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation After Stimulation Using CFSE Staining and Flow Cytometry

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphpoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
The cell cycle is a universal process of life. During the cell cycle, a cell undergoes several modifications to divide into two daughter cells. This mechanism occurs throughout an organism&#39;s life in response to its needs. Cell divisions and embryonic development produce a full organism from a single-celled zygote. During adulthood, the cell cycle is central to many critical biological processes, such as tissue repairs.
Mechanisms of cell division are tightly controlled events where the cell undergoes stepwise modifications before final division. Cells that are not yet in the cycle are described as being in the Gap 0 (G0) phase. During this stage the cell is considered quiescent. When the cell starts to cycle, four distinct phases are recognized: Gap 1 (G1), Synthesis (S), Gap 2 (G2) and Mitosis (M). G1 phase is a checkpoint for resources needed by the cell for DNA synthesis. Then, S phase occurs, and DNA replication starts, followed by the G2 interphase, another checkpoint that controls all elements necessary to for the cell to divide. Finally, the cell enters mitosis and divides into two daughter cells.
Cell division is a highly informative parameter in many different biological systems. In the field of immunology, analysis of leukocyte proliferation can indicate the mechanism of the immune response. Other domains of investigation also rely on cell cycle analysis. For instance, analysis of the cell cycle during tumor development has improved our understanding of cancer.
Many fluorescent dyes are now available for tracking cell proliferation. These dyes differ in their chemical and spectral properties. Two different classes of dyes exist: protein dyes permanently combine with protein by forming a covalent bond, and membrane dyes stably intercalate within cell membranes via strong hydrophobic associations. In vitro and in vivo studies of immune cell proliferation by flow cytometry are among the most common applications of both classes of cell tracking dyes (1, 2).
CFSE (Carboxyfluorescein succinimidyl ester) is a fluorescent dye that marks dividing cells. Initially, all the cells receive the same amount of dye; dividing cells evenly split the dye they received between their two daughter cells. Consequently, the cell cycle can be followed by the progressive decrease of dye intensity in the cells. CFSE staining is followed by conventional multi-parametric flow cytometry, a high-throughput, fluorescence-based technology that allows phenotypic and functional characterization of cells based on their degree of CFSE staining (3).
In the following experiment, we assess the proliferation of CD4+ and CD8+ T cells in-vitro, following CD3 stimulation, using CFSE staining and flow cytometry.

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation

细胞周期分析:使用CFSE染色和流细胞测定法评估刺激后CD4和CD8 T细胞增殖

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3
1 Unit for Lymphpoiesis, Department of Immunology, Pasteur ...

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Bromodeoxyuridine Pulse Labelling Assay: A Technique to Measure Cell Proliferation

Source: Welschinger R. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/52840">Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization.</a> J. Vis. Exp. (2015)
The video describes the stepwise pulse labeling assay of mammalian cells with bromodeoxyuridine (BrdU) for measuring cell proliferation. The BrdU uptake permits the temporal tracking of cells that were in the synthesis phase at a specific point in time, without requiring the cells to be synchronized.

溴脱氧尿嘧啶脉冲标记测定:一种测量细胞增殖的技术

Source: Welschinger R. et al. Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization. J. Vis. Exp. (2015)
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细胞周期进程的时间跟踪使用流式细胞仪，无需同步

摘要

探索更多视频

此视频中的章节

通过两个互补细胞同步方案研究细胞周期调控的基因表达

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

基于同步成像和流式细胞术检测治疗诱导的衰老癌细胞中的多种荧光衰老标志物

通过细胞周期激酶和磷酸酶活性成比例的FRET监测

在哺乳动物细胞中细胞周期位置分析

代谢标记和流式细胞仪测定细胞周期进程的动力学

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

通过流式细胞术同时评估造血干细胞和祖细胞的亲缘关系、分裂数和表型

细胞周期分析:使用CFSE染色和流细胞测定法评估刺激后CD4和CD8 T细胞增殖

溴脱氧尿嘧啶脉冲标记测定:一种测量细胞增殖的技术

细胞周期进程的时间跟踪使用流式细胞仪，无需同步

摘要

探索更多视频

此视频中的章节

通过两个互补细胞同步方案研究细胞周期调控的基因表达

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

基于同步成像和流式细胞术检测治疗诱导的衰老癌细胞中的多种荧光衰老标志物

通过细胞周期激酶和磷酸酶活性成比例的FRET监测

在哺乳动物细胞中细胞周期位置分析

代谢标记和流式细胞仪测定细胞周期进程的动力学

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

通过流式细胞术 同时 评估造血干细胞和祖细胞的亲缘关系、分裂数和表型

细胞周期分析:使用CFSE染色和流细胞测定法评估刺激后CD4和CD8 T细胞增殖

溴脱氧尿嘧啶脉冲标记测定:一种测量细胞增殖的技术

通过流式细胞术同时评估造血干细胞和祖细胞的亲缘关系、分裂数和表型