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/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/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 border="0" cellpadding="0" cellspacing="0">
	<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 
			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-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 1640 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&#8242;-deoxyuridine</td>
			<td>Sigma</td>
			<td>B5002-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon TC 150cm2 vented Flasks</td>
			<td>BD Biosciences</td>
			<td>355001</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes 25mL</td>
			<td>Greiner</td>
			<td>760180</td>
			<td></td>
		</tr>
		<tr>
			<td>Aersol Pipettes 200&#181;L</td>
			<td>Interpath</td>
			<td>24700</td>
			<td></td>
		</tr>
		<tr>
			<td>Aersol Pipettes 1mL</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>CO2 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><a name="RANGE!C23">2348S</a></td>
			<td></td>
		</tr>
		<tr>
			<td>NALM6&#160;</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， ...

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

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

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 ...

temporal-tracking-cell-cycle-progression-using-flow-cytometry-without

Research

Education

JoVE Journal

JoVE Core

Biology

Cellular Processes

Cell Cycle and Division

SCIENTISTS IN ACTION

19.0K 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

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

科研

生物学

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 ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

在斑马鱼发育的细胞追踪使用Photoconvertible蛋白

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

对于外小泡的使用流式细胞仪分析技术

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

同步<em&gt;新月柄杆菌</em&gt;对细菌细胞周期的研究

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

LED Thermo Flow &amp;#8212; Combining Optogenetics with Flow Cytometry

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

LED热流量 - 结合光遗传学与流式细胞仪

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

重组离子通道的相对细胞表面表达的总用流式细胞仪测定

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

有丝分裂细胞同步与高分辨共聚焦显微镜联合应用研究细胞周期蛋白在有丝分裂过程中的作用

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...