这项工作得到了 NIH CA109821 的支持。我们感谢贝克曼-库尔特慷慨提供资金, 以支付出版费用和在淘洗系统的设置和运作方面的技术援助。我们感谢 Dr. 弗雷德 Falkenburg 提供初始的主要的所有文化。

注意: 此协议已针对主要 B-所有单元格进行了优化, 其直径范围从大约8和 #181; m (G1 阶段) 到13和 #181; m (G2/M 阶段)。因此, 具体的离心机速度和泵流量可能不适用于不同尺寸范围的电池。然而, 利用沉降速率方程可以估计适当的淘洗参数。细胞在一个被定义的无血清培养基被培养, 如描述的 16 在优选的密度 1-3 x 10 6 细胞/毫升。除4和 #176 所进行的馏分的收集外; c 使用冰冷管, 所有步骤都在室温下进行, 离心机设置在20和 #176; c 的最大值为24和 #176; c. 
 1. 准备淘洗缓冲和材料 <ol> <li> 为了减少细胞的结块, 准备一个淘洗的缓冲液和 #39 的平衡盐水溶液 (HBSS) 与酚红补充2% 胎牛血清 (FBS), 保护细胞免受机械应力在淘洗过程中, 和1.6 克/升的 2-萘酚-68 二磺酸二钾盐 (NDA), 这使得细胞的外部负电荷和减少结块。
	<ol> <li> 向50毫升锥形管添加1.6 克 NDA. </li>
		<li> 在无菌条件下, 将35毫升的 HBSS 从1升瓶中添加到包含 NDA 的50毫升圆锥管中。漩涡管, 直到 NDA 已解散. </li>
		<li> 使用0.2 和 #181; m 过滤器, 将溶解的 NDA 转移到新的无菌50毫升锥形管中。然后将 NDA 添加到其余的 HBSS 1 L. </li>
		<li> 最后, 向 HBSS 添加21毫升 FBS, 以获得最终浓度为 2% (v/v). 
		注: 淘洗缓冲器可以准备和存储在4和 #176; C 直到截止日期在 HBSS 或直到有一个主要的变化, 在 pH 值表明, 由指示染料泛黄. </li>
	</ol> </li> <li> 标签50毫升锥形管收集的分数, 使有管标记洗涤1和 2 (W1 和 W2), 分数 1-20 (F1 和 #160;-F20), 并停止。
	<ol> <li> 在收集分数之前先在冰上冷却这些管子. </li>
	</ol> </li> </ol> 2。淘洗系统的程序集 <ol> <li> 首先, 将频闪组件安装到离心机中, 使电源线通过左侧的端口送入会议厅。确保闪光灯闪灯在房间的前面, 这样当离心机关闭时, 它会与观察窗口排成一线。
	<ol> <li> 将两个梅花头螺钉拧紧到每个托架顶部的两个孔中, 然后拧紧每个托架底部的螺钉, 以确保固定组件的安全. </li>
		<li> 将电源线插入离心机背面的频闪电源端口. </li>
	</ol> </li> <li> 下一步, 将转子垂直向下设置到离心机驱动器集线器上, 并使用 T 型手柄的六角扳手进行安全处理. 
	注: 如果离心机不需要为 non-elutriation 目的, 闸门组件和转子可以保持在离心机之间的运行. </li>
	<li> 一旦转子是固定的, 放置的快速释放组件包含淘洗室, 计数器平衡, 旋转密封组件, 和安装板进入转子。
	<ol> <li> 用一只手在淘洗腔上均匀向下推, 另一只手放在计数器的天平上, 直到转子上的螺栓进入安装板上的孔. </li>
		<li> 最后, 通过将电缆一侧的针脚放入旋转密封组件的孔中, 并将电缆的另一侧的 eyehole 固定在左侧托架上的一个十字头螺钉 (2.1.1 中提到) 上, 从而连接锚定电缆。f 会议厅. </li>
	</ol> </li> <li> 下一步, 使用变速泵和油管组装流系统, 将缓冲器泵入淘洗腔, 并在淘洗室流出时收集缓冲器。
	<ol> <li> 将大小为16的输入管插入来自变速泵, 通过在离心室左上方的端口, 使其进入会议厅. </li>
		<li> 将管接头连接到输入管的自由端, 并将管件插入旋转密封组件一侧的输入孔中. </li>
		<li> 通过端口插入尺寸为16的输出管, 并将其连接到旋转密封组件顶部的传输管上. 
		注: 流系统还可以在淘洗运行之间保持组装. </li>
	</ol> </li> <li> 最后, 打开离心机并设置实验所需的规格。
	<ol> <li> 更改转子 ID. </li>
		<li> 如果离心机需要时间输入, 则设置为2小时, 这足以完成淘洗运行. </li>
		<li> 将温度设置为20和 #176; c 的最大 temp 为24和 #176; c. </li>
		<li> 设置最大加速度和减速速度. </li>
		<li> 最后, 将速度设置为 867 x g. </li>
	</ol> </li> </ol> 3。淘洗系统的启动 <ol> <li> 首先为了对淘洗系统进行消毒, 将储油管放入500毫升的塑料瓶中, 其中含有5% 漂白剂, 并将输出管放入一个空的 1 L 塑料废物瓶中。
	<ol> <li> 打开泵并将速度设置为25毫升/分钟, 并允许5% 漂白剂流经淘洗系统, 直到它从输出管泵出到垃圾桶中为止. </li>
		<li> 打开离心机, 使其达到最大指定速度 (867 x g), 以释放气泡和反压. </li>
		<li> 停止离心机。一旦转子停止旋转, 打开盖子和检查室的任何泄漏. </li>
		<li> 如果没有泄漏, 让漂白剂溶液流经系统至少5分钟. </li>
	</ol> </li> <li> 下一步, 用蒸压水冲洗系统, 因为漂白剂可以导致从淘洗缓冲中形成沉淀物, 这可能导致细胞结块, 并对细胞造成伤害。
	<ol> <li> 将储油管管放入一瓶1升的蒸压水中, 并允许其冲洗至少10分钟. </li>
	</ol> </li> <li> 用水冲洗后, 将淘洗缓冲区引入系统。使用一个150毫升的玻璃瓶, 已被蒸压, 以创建一个水库的淘洗缓冲和细胞。
	<ol> <li> 将100毫升的淘洗缓冲液添加到瓶子中, 并将储油管移到玻璃瓶中, 使管道处于最底部. </li>
		<li> 启动离心机并允许缓冲区流经系统以冲洗水. </li>
		<li> 一旦储气瓶几乎是空的, 并且水从系统中完全冲出, 将输出管从废物瓶移到蓄水池, 以便通过系统回收缓冲液. </li>
	</ol> </li> <li> 最后, 调整查看窗口, 以便在当前速度下可以看到淘洗室。
	<ol> <li> 按离心机外部的频闪灯电源按钮, 使灯打开。转淘洗 k也在离心机的外部, 一路向左. </li>
		<li> 在查看窗口时, 慢慢地将旋钮向右转, 直到淘洗室进入视野, 并且闪光灯与转子的旋转时间有关. 
		注意: 淘洗系统可以保留在此状态, 直到单元格准备就绪. </li>
	</ol> </li> </ol> 4。制备细胞样本 <ol> <li> 将生长培养基中的细胞数和分300到 4亿, 放入几个50毫升的锥形管中。旋转 1210 x g 的细胞3分钟. </li>
	<li> 在25毫升的淘洗缓冲液中, 通过移轻轻地向上和向下吸出生长培养基和重细胞. </li>
	<li> 为了减少细胞结块, 通过一个25口径的针通过两次细胞。
	<ol> <li> 使用10毫升注射器绘制单元格, 然后连接25针, 并将电池通过到无菌50毫升锥形管的内侧壁。重复这一点, 直到所有25毫升通过针. </li>
		<li> 获得一个新的针头和注射器, 并重复上述过程一次. 
		注意: 注射器针应该放在锋利的容器里. 
		注意: 在将细胞引入淘洗系统之前, 必须立即通过针头, 使细胞聚集到最小. </li>
	</ol> </li> </ol> 5。将细胞样本引入淘洗系统和分数的收集 <ol> <li> 通过将单元格样本注入到具有剩余淘洗缓冲区回收的储罐中, 将单元格引入淘洗系统中.
	<ol> <li> 让单元格进入系统并在淘洗腔内达到平衡. 
		注意: 这一过程大约需要5分钟, 通过观察窗口进入会议厅, 可以跟踪这些细胞的进展情况。为了确保所有的细胞都在室内, 采取一滴淋洗, 放置在一个玻璃幻灯片, 并在显微镜下查看。如果样品没有完整的细胞, 这证实了它们在腔室中的滞留. </li>
	</ol> </li> <li> 开始收集分数一旦所有的细胞进入会议厅, 并有一个明确的边界, 在最广泛的部分, 其中细胞处于平衡。
	<ol> <li> 首先将输出管移动到标记为 W1 的 50 mL 圆锥管上。收集50毫升的淘洗缓冲区的这一分数, 确保保持一个眼睛的油箱和补充淘洗缓冲必要时. 
		注: 为了确保所有的细胞都进入了系统, 让储罐在第一次充注水库之前变得几乎是空的。第一次灌装后, 将不必等待水库变得几乎空。此外, 永远不要让坦克变成空, 因为这将产生气泡和背部压力在系统中. </li>
		<li> 一旦50毫升已收集 W1, 同时改变速度到 821 x g 和移动输出管的锥形管标记 W2 和收集50毫升以上的速度. </li>
		<li> 继续更改速度和收集分数, 如表1所示。确保储罐中有介质, 并始终保持锥形管在冰上。最后, 当速度减小时, 通过缓慢地将旋钮向右旋转来调整查看窗口. </li>
		<li> 一旦所有20分数被收集, 停止离心机和收集50毫升在锥形管标记停止. </li>
	</ol> </li> </ol> 6。淘洗系统的冲洗 <ol> <li> 在离心机停止完全冲洗系统后, 将蓄水池管放在1升的蒸压水中, 并将输出管放回废瓶中。
	<ol> <li> 将泵速提高到50毫升/分钟, 并允许水通过系统运行, 直到耗尽为止. </li>
	</ol> </li> <li> 关闭泵, 从快速释放组件中卸下输入和输出软管, 然后从锚索拔下针脚。
	<ol> <li> 从转子上卸下快速释放组件. 
		注: 根据离心机的用途, 淘洗系统现在可以离开了。否则, 整个程序集可以按上述顺序进行相反的排序。淘洗室可从快速释放组件中取出, 并在使用超声波水浴的运行之间进行清洗。这将清除可能对以后运行产生负面影响的腔壁上的碎片和污染物. </li>
	</ol> </li> </ol> 7。分析 G1 或 G2/M 池的分数和集合. 
 注意: 为了证明淘洗的有效性, 每个分数都被分析为细胞计数、细胞直径和 DNA 含量, 而池是在 G1 或 G2/M 阶段为免疫分析创建的细胞, 如代表性的结果。本协议部分将介绍如何创建这些池并准备它们以供实验使用. 
<ol> <li> 将每个分数在 2000 x g 处旋转5分钟, 在4和 #176; C 和吸气淘洗缓冲. </li>
	<li> 将分数组合在一起以获得 G1 相池和 G2/M 阶段池。
	<ol> <li> 对于 G1 相池, 重的分数在 788 x g 到 661 x g (F1-F5) 的速度范围内收集, 总共1毫升磷酸盐缓冲盐水溶液 (PBS), 并转移到15毫升锥形管。对于 G2/M 相池, 在 387 x g 和 333 x g (F17-F20) 的速度范围内收集的重分数. </li>
		<li> 在4和 #176 上向下旋转 2000 x g 处的单元格, 并从 PBS 中抽离. </li>
	</ol> </li> <li> 在10毫升的生长介质中重单元格并进行单元格计数. </li>
	<li> 从每个池中提取一个分, 通过碘碘化染色和流细胞分析来分析 DNA 含量 15 . 
	注: 每个池的剩余部分可用于进一步的实验. </li>
</ol>

<ol>
	<li>Beach, D., Piper, M., Shall, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+newly-initiated+DNA+from+the+early+S+phase+of+the+synchronous+eukaryote,+Physarum+polycephalum.">Isolation of newly-initiated DNA from the early S phase of the synchronous eukaryote, Physarum polycephalum.</a> Exp cell res. 129, 211-221 (1980).</li><li>Sevcikova, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Completion+of+cell+division+is+associated+with+maximum+telomerase+activity+in+naturally+synchronized+cultures+of+the+green+alga+Desmodesmus+quadricauda.">Completion of cell division is associated with maximum telomerase activity in naturally synchronized cultures of the green alga Desmodesmus quadricauda.</a> FEBS letters. 587, 743-748 (2013).</li><li>Rosner, M., Schipany, K., Hengstschlager, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merging+high-quality+biochemical+fractionation+with+a+refined+flow+cytometry+approach+to+monitor+nucleocytoplasmic+protein+expression+throughout+the+unperturbed+mammalian+cell+cycle.">Merging high-quality biochemical fractionation with a refined flow cytometry approach to monitor nucleocytoplasmic protein expression throughout the unperturbed mammalian cell cycle.</a> Nat protoc. 8, 602-626 (2013).</li><li>Banfalvi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+cell+synchronization.">Overview of cell synchronization.</a> Meth mol biol. 761, 1-23 (2011).</li><li>Langan, T. J., Rodgers, K. R., Chou, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+Mammalian+Cell+Cultures+by+Serum+Deprivation.">Synchronization of Mammalian Cell Cultures by Serum Deprivation.</a> Meth mol biol. 1524, 97-105 (2017).</li><li>Dulla, K., Margalef, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-Scale+Mitotic+Cell+Synchronization.">Large-Scale Mitotic Cell Synchronization.</a> Meth mol biol. 1524, 65-74 (2017).</li><li>Siefert, J. C., Clowdus, E. A., Sansam, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+control+in+the+early+embryonic+development+of+aquatic+animal+species.">Cell cycle control in the early embryonic development of aquatic animal species.</a> Comp biochem physiol Toxicol,pharmacol CBP. 178, 8-15 (2015).</li><li>Banfalvi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+synchronization+of+animal+cells+and+nuclei+by+centrifugal+elutriation.">Cell cycle synchronization of animal cells and nuclei by centrifugal elutriation.</a> Nat protoc. 3, 663-673 (2008).</li><li>Grosse, J., Meier, K., Bauer, T. J., Eilles, C., Grimm, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+separation+by+countercurrent+centrifugal+elutriation:+recent+developments.">Cell separation by countercurrent centrifugal elutriation: recent developments.</a> Prep biochem,biotechnol. 42, 217-233 (2012).</li><li>Smith, J., Manukyan, A., Hua, H., Dungrawala, H., Schneider, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+Yeast.">Synchronization of Yeast.</a> Meth mol. 1524, 215-242 (2017).</li><li>Gascoigne, K. E., Taylor, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+anti-mitotic+drugs+kill+cancer+cells?.">How do anti-mitotic drugs kill cancer cells?.</a> J cell sci. 122, 2579-2585 (2009).</li><li>Manchado, E., Guillamot, M., Malumbres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Killing+cells+by+targeting+mitosis.">Killing cells by targeting mitosis.</a> Cell death diff. 19, 369-377 (2012).</li><li>Komlodi-Pasztor, E., Sackett, D., Wilkerson, J., Fojo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitosis+is+not+a+key+target+of+microtubule+agents+in+patient+tumors.">Mitosis is not a key target of microtubule agents in patient tumors.</a> Nature reviews. Clinic oncol. 8, 244-250 (2011).</li><li>Furst, R., Vollmar, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+perspective+on+old+drugs:+non-mitotic+actions+of+tubulin-binding+drugs+play+a+major+role+in+cancer+treatment.">A new perspective on old drugs: non-mitotic actions of tubulin-binding drugs play a major role in cancer treatment.</a> Die Pharmazie. 68, 478-483 (2013).</li><li>Kothari, A., Hittelman, W. N., Chambers, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Cycle-Dependent+Mechanisms+Underlie+Vincristine-Induced+Death+of+Primary+Acute+Lymphoblastic+Leukemia+Cells.">Cell Cycle-Dependent Mechanisms Underlie Vincristine-Induced Death of Primary Acute Lymphoblastic Leukemia Cells.</a> Cancer res. 76, 3553-3561 (2016).</li><li>Nijmeijer, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+culture+of+primary+human+lymphoblastic+leukemia+cells+in+the+absence+of+serum+or+hematopoietic+growth+factors.">Long-term culture of primary human lymphoblastic leukemia cells in the absence of serum or hematopoietic growth factors.</a> Exp hematol. 37, 376-385 (2009).</li><li>Knudsen, E. S., Wang, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+mechanisms+for+the+inhibition+of+E2F+binding+to+RB+by+cyclin-dependent+kinase-mediated+RB+phosphorylation.">Dual mechanisms for the inhibition of E2F binding to RB by cyclin-dependent kinase-mediated RB phosphorylation.</a> Mol Cell Bio. 17, 5771-5783 (1997).</li><li>Malumbres, M., Barbacid, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cyclin-dependent+kinases.">Mammalian cyclin-dependent kinases.</a> Trends biochem sci. 30, 630-641 (2005).</li><li>Liang, H., Hittelman, W., Nagarajan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitous+expression+and+cell+cycle+regulation+of+the+protein+kinase+PIM-1.">Ubiquitous expression and cell cycle regulation of the protein kinase PIM-1.</a> Arch biochem biophys. 330, 259-265 (1996).</li><li>Sen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+gene+encoding+a+tRNA+synthetase-like+protein+is+enhanced+in+tumorigenic+human+myeloid+leukemia+cells+and+is+cell+cycle+stage-+and+differentiation-dependent.">Expression of a gene encoding a tRNA synthetase-like protein is enhanced in tumorigenic human myeloid leukemia cells and is cell cycle stage- and differentiation-dependent.</a> Proc Natl Acad Sci USA. 94, 6164-6169 (1997).</li><li>Ly, T., Endo, A., Lamond, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+the+response+to+cell+cycle+arrests+in+human+myeloid+leukemia+cells.">Proteomic analysis of the response to cell cycle arrests in human myeloid leukemia cells.</a> eLife. 4, (2015).</li></ol>

妮米隆·科塔里目前在 St. 裘德儿童研究医院的细胞和分子生物学系。

我们描述了使用 CCE 在细胞周期的不同阶段获得主所有细胞的方法。在最佳条件下, G1 相中的细胞的基本纯种群和 G2/M 期细胞的高度丰富的种群可以很容易地获得优良的产量, 如果需要的话, 在 S 相中高度丰富的细胞也可以得到。这里提出的结果是使用一个主要的所有文化 (特别是, ALL-5), 并取得了基本上相同的结果, 与独立的文化, ALL-215。另外, 所有其他主要的所有文化在异步增长?...

培养细胞通常以异步方式生长, 而单个细胞则存在于细胞周期的不同阶段。有些有机体自然地陈列同步细胞周期或可以由具体生理刺激同步。例如, 在粘液霉菌的巨型疟原虫中的细胞核,多头多头在高度同步的方式下进行了1, 绿藻的细胞Desmodesmus quadricauda可以通过交替的光和暗来同步期间2。虽然这些生物体提供了独特的实验特性, 但它们没有充分地模拟哺乳动物细胞的复杂性。人工同步哺乳动物细胞的能力一直是促进我们对细胞周期调节和细胞周期检查点的分子基础的理解的中心。常见的方法包括血清剥夺, 化学阻滞和释放, 或利用物理特性3,4。血清的提取经常导致细胞进入平静, 并且血清的 re-addition 促进细胞周期再入进入 G1 阶段5。化学抑制剂包括诸如过多的胸苷或羟基的药物, 这些因子阻断了 G1/S 边界的细胞, 或通常在 M 阶段34中逮捕细胞的微管抑制剂。利用物理特性的方法包括有丝分裂 shake-off, 它可以用来丰富有丝分裂细胞, 因为它们比相间细胞的黏附性小6。然而, 所有这些技术都有潜在的缺点。例如, 并非所有的细胞类型在缺乏血清或化学抑制剂存在的情况下都保持生存能力, 有丝分裂 shake-off 后细胞的收率是有限的, 不需要事先与有丝分裂抑制剂同步。
除了早期的胚胎细胞周期, 当细胞分裂为7时, 它们的大小逐渐减小, 但大多数细胞在循环中前进, 经历了生长阶段, 变得更大。此属性在逆流离心淘洗 (CCE) 技术中被利用, 它可用于分隔大小不同的单元格, 从而在单元周期阶段8,9中分离。在 CCE 期间, 细胞受到两种相反的作用力的影响: 离心力, 它驱动细胞远离自转轴, 流体速度 (逆流), 它驱动细胞朝向旋转轴 (图 1)。在淘洗室的细胞达到平衡的位置, 这些力量是平等的。支配平衡位置的关键因素是细胞直径和密度。随着缓冲液流速的增加, 逆流阻力大于离心力, 建立了新的平衡, 引起腔内细胞位置的变化。所有的细胞都被转移到腔室的出口, 导致较小的单元先离开腔室, 而较大的细胞则留在腔室内, 直到逆流率增加, 以促进其退出。淘洗室逃逸的细胞, 连续增加逆流率, 可以收集在特定的分数和每个分数包含按顺序增加大小的细胞。随着逆流速率的增加, 离心速度的下降会导致离心力的连续下降, 而不是淘洗。淘洗的过程需要根据细胞类型、使用的淘洗缓冲器和所用的特定仪器进行优化。在这些条件下, 细胞沉淀的速率最好用斯托克斯定律描述: sv = [d2(ρp-ρm)/18η]. ω2r, 其中 sv = 沉淀速度;d = 微粒的直径;ρp = 粒子的密度;ρm = 缓冲区的密度;η = 缓冲器的粘度;ω = 转子的角速度;和 r = 粒子的径向位置。因此, SV 与细胞直径和密度成正比, 因为直径被提高到第二个功率, 它的贡献比密度通常是通过细胞周期常数。
CCE 的一个重要优点是, 细胞不受化学治疗或营养缺乏的苛刻条件的影响, 可以在良好的产量中恢复, 基本上是泰然自若的。一个要求是, 当它们遍历细胞周期时, 所讨论的细胞直径会显著增加, 至少有30%。主要缺点是专用离心机、转子和附件的成本。尽管如此, 通过 CCE 培养细胞在特定细胞周期阶段富集的能力, 极大地促进了细胞周期研究从酵母到哺乳动物细胞9,10。此外, 最近的进展是扩大用于分离的细胞从健康与癌组织, 分离不同的细胞类型在异构混合物, 并生产特定的细胞类型的免疫治疗的9。
我们的主要兴趣是了解微管靶向剂 (mta) 的作用机制, 如长春生物碱和紫杉。这些药物被认为完全是在有丝分裂, 阻断纺锤微管功能11,12, 但最近的证据表明它们也可能靶向界面微管13,14。我们最近报告说, 原发性急性淋巴细胞白血病 (所有) 细胞在 G1 期或 M 期发生死亡时, 以长春新碱和其他 mta 的细胞周期依赖性的方式处理15。通过 CCE 将所有细胞分离成不同的细胞周期阶段的能力有助于达到这个结论。在本文中, 我们描述的技术细节, 并提供实用的技巧, CCE 的应用, 以准备在不同的细胞周期阶段的主所有细胞。

<table border="0" cellpadding="0" cellspacing="0" width="1542">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">Hanks&#39; balanced salt solution</td>
			<td style="width:108px;">Lonza</td>
			<td style="width:119px;">10-508Q</td>
			<td style="width:780px;">1 L bottle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">Fetal Plus bovine serum</td>
			<td style="width:108px;">Atlas Biologicals</td>
			<td style="width:119px;">FP-0500-A</td>
			<td>500 mL bottle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">2-napthol-6,8-disulfonic acid dipotassium salt</td>
			<td style="width:108px;">Acros Organics</td>
			<td style="width:119px;">212-672-4</td>
			<td>100 g powder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">50 mL concial tubes</td>
			<td style="width:108px;">Denville Scientifics</td>
			<td style="width:119px;">C1062-P</td>
			<td>500/case</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">PES Membrane 0.22 &#181;m filter unit</td>
			<td style="width:108px;">Millipore</td>
			<td style="width:119px;">SLGP033RB</td>
			<td>250/pack</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Avanti J-26S XPI w/ Elut. Non-IVD - 50/60 Hz, 200/208/240V</td>
			<td style="width:108px;">Beckman Coulter</td>
			<td style="width:119px;">B14544</td>
			<td>centrifuge compatible with elutriation system</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">JE 5.0 elutriator rotor kit</td>
			<td style="width:108px;">Beckman Coulter</td>
			<td style="width:119px;">356900</td>
			<td>rotor kit which includes the rotor, T-handle hex wrench, the quick release assembly (w/o the elutriation chamber), anchoring cable, and the strobe assembly</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Chamber, standard, 4-mL, &#34;A&#34;</td>
			<td style="width:108px;">Beckman Coulter</td>
			<td style="width:119px;">356943</td>
			<td>standard elutriation chamber&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">Masterflex L/S Easy-Load pump head</td>
			<td style="width:108px;">Cole-Parmer</td>
			<td style="width:119px;">EW-07518-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">Masterflex L/S Variable-Speed Drive</td>
			<td style="width:108px;">Cole-Parmer</td>
			<td style="width:119px;">EW-07528-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">Masterflex BioPharm platinum-cured silicone pump tubing, L/S 16</td>
			<td style="width:108px;">Cole-Parmer</td>
			<td style="width:119px;">EW-96420-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">25 G x 1.5 Precision Glide needle</td>
			<td style="width:108px;">BD</td>
			<td style="width:119px;">305127</td>
			<td>sterile, single-use needles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">10 mL Luer-Lok syringe</td>
			<td style="width:108px;">BD</td>
			<td style="width:119px;">309604</td>
			<td>sterile, single-use syringes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">Vi-Cell XR</td>
			<td style="width:108px;">Beckman Coulter</td>
			<td style="width:119px;">383556</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">PI/RNase staining buffer</td>
			<td style="width:108px;">BD Pharmingen</td>
			<td style="width:119px;">550825</td>
			<td>propidium iodide/RNase staining buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">cyclin B1 (GNS1) mouse monoclonal IgG antibody</td>
			<td style="width:108px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-245</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">cyclin D1 (DCS-6) mouse monoclonal IgG antibody</td>
			<td style="width:108px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-20044</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">P-Rb (S807/811) (D20B12) XPR rabbit monoclonal antibody</td>
			<td style="width:108px;">Cell Signaling Technology</td>
			<td style="width:119px;">8516s</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">GAPDH (14C10) rabbit monoclonal antibody</td>
			<td style="width:108px;">Cell Signaling Technology</td>
			<td style="width:119px;">2118s</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">Goat anti-mouse IgG (H+L)-HRP conjugate</td>
			<td style="width:108px;">BioRad</td>
			<td style="width:119px;">170-6516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">Goat anti-rabbit IgG (H+L)-HRP conjugate</td>
			<td style="width:108px;">BioRad</td>
			<td style="width:119px;">170-6515</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of primary acute lymphoblastic leukemia cells in different cell cycle phases by centrifugal elutriation

同步细胞的能力一直是促进我们对细胞周期调节的理解的中心。常用的技术包括血清剥夺;在不同的细胞周期阶段逮捕细胞的化学物质;或使用有丝分裂 shake-off, 利用其减少粘附。然而, 所有这些都有缺点。例如, 血清饥饿对正常细胞很有效, 但对于因癌基因活化或肿瘤抑制丧失而受损的细胞周期检查点的肿瘤细胞来说, 效果却不那么好。同样地, 经过化学处理的细胞群可以产生药物引起的损伤, 并显示与压力相关的改变。绕过这些问题的一种技术是逆流离心淘洗 (CCE), 在这里, 细胞受到两种相反的作用力, 离心力和流体速度, 导致细胞在大小和密度的基础上分离。由于细胞通过周期的推进通常会扩大, CCE 可用于将细胞分离成不同的细胞周期阶段。我们将此技术应用于原发性急性淋巴细胞白血病细胞。在最佳条件下, G1 期细胞的基本纯种群和 G2/M 期细胞的高富集率可获得优良的产量。这些细胞的数量非常适合研究抗癌药物和其他应用的细胞周期依赖性机制。我们还展示了对标准过程的修改如何导致次优的性能, 并讨论了技术的局限性。所提出的详细方法应有助于对其他类型的细胞进行技术的应用和探索。

主所有单元格 (3-4 x 108) 受离心淘洗, 并收集到两个洗涤分数和二十主要分数, 如协议中所述。表1显示了代表性的数据, 其中每一个部分的细胞总数, 以及相应的转子速度提出。总体产量一般在80% 以上。单个分数中细胞直径的测量证实了细胞直径随推进淘洗而增加 (图 2)。这些结果代表了两个独立确定的平均值, 反映了数据重现性高的程度。分?...

Watch this Scientific Journal Video about Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation at JoVE.com

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

本议定书描述了使用离心淘洗分离原发性急性淋巴细胞白血病细胞到不同的细胞周期阶段。

离心淘洗制备不同细胞周期的原发性急性淋巴细胞白血病细胞

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

preparation-primary-acute-lymphoblastic-leukemia-cells-different-cell

Research

JoVE Journal

Cancer Research

8.2K 次观看.  University of Arkansas for Medical Sciences. 本议定书描述了使用离心淘洗分离原发性急性淋巴细胞白血病细胞到不同的细胞周期阶段。

视频: Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

测定急性髓系白血病可测残留病和白血病干细胞的骨髓标本和过程综合协议

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

科研

癌症研究

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in adults. Initially, AML is a disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of leukemia blast cells, and reduced production of functional hematopoietic elements. Heterogeneity extends to the presence of leukemia stem cells (LSC), with this dynamic cell compartment evolving to overcome various selection pressures imposed upon during leukemia progression and treatment. To further define the LSC population, the addition of CD90 and CD45RA allows the discrimination of normal HSCs and multipotent progenitors within the CD34+CD38- cell compartment. Here, we outline a protocol to detect simultaneous expression of several putative LSC markers (CD34, CD38, CD45RA, CD90) on primary blast cells of human AML by multiparametric flow cytometry. Furthermore, we show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts. This method of detection and quantification of putative LSC may be used for clinical follow-up of chemotherapy response (i.e., minimal residual disease), as residual LSC may cause AML relapse.

We outline a protocol to detect simultaneous expression of leukemia stem cell markers on primary acute myeloid leukemia cells by flow cytometry. We show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts.

流式细胞仪估测原发性急性髓系白血病和患者源性移植中白血病干细胞的诊断和随访

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly indicate that aneuploidy triggers genome instability, ultimately generating cells with complex karyotypes that arrest their proliferation. Isolation and characterization of cells harboring complex karyotypes are crucial to study the impact of an imbalanced chromosome number on cell physiology. To date, no methods have been established to reliably isolate such aneuploid cells. This paper provides a protocol for the enrichment and analysis of aneuploid cells with complex karyotypes utilizing standard, inexpensive tissue culture techniques. This protocol can be used to analyze several features of aneuploid cells with complex karyotypes including their induced senescence-associated secretory phenotype, pro-inflammatory properties, and ability to interact with immune cells. Because cancer cells often harbor imbalances in chromosome number, it is crucial to decipher how aneuploidy impacts cell physiology in normal cells, with the ultimate goal of uncovering both its pro- and anti-tumorigenic effects.

Aneuploidy leads to genome instability, which eventually produces cell cycle-arrested cells with complex karyotypes. This paper provides a simple and convenient method to isolate aneuploid cells with complex karyotypes that cease to divide.

复杂核型细胞周期捕获细胞的生成与分离

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

Assessment of the Metabolic Profile of Primary Leukemia Cells

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

原发性白血病细胞代谢谱的评价

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

木尿造血干细胞和祖细胞和MLL-AF9驱动白血病线粒体反应氧物种的流细胞学分析

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

建立胃癌患者衍生异种移植模型和原细胞系

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

免疫荧光对非小细胞肺癌患者循环肿瘤细胞的半自动PD-L1特征化及枚举

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

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 ...

Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant cellular localization disrupting their mechanism of action. The survival of chronic lymphocytic leukaemia (CLL) cells, among other cancer cells, is assisted by the deregulation of nuclear to cytoplasmic shuttling, at least in part through deregulation of the transport receptor XPO1 and the constitutive activation of PI3K-mediated signaling pathways. It is essential to understand the role of individual proteins in the context of their intracellular location to gain a deeper understanding of the role of such proteins in the pathobiology of the disease. Furthermore, identifying processes that underlie cell stimulation and the mechanism of action of specific pharmacological inhibitors, in the context of subcellular protein trafficking, will provide a more comprehensive understanding of the mechanism of action. The protocol described here enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These fractions can be used to determine changes in protein trafficking between the nuclear and cytoplasmic fractions upon cell stimulation and drug treatment. The data can be quantified and presented in parallel with immunofluorescent images, thus providing robust and quantifiable data.

This protocol enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These samples are used to determine protein localization as well as changes in protein trafficking that take place between the nuclear and cytoplasmic compartments upon cell stimulation and drug treatment.

原发性慢性淋巴细胞白血病细胞的细胞次细胞分段监测核/细胞质蛋白贩运

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant ...

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties (so called leukemic stem cells, LSCs) from more differentiated counterpart leukemic cells that lack disease initiation potential although they carry similar leukemia specific genetic mutations. NKG2DL are biochemically highly diverse MHC class I-like self-molecules. Healthy cells in homeostatic conditions generally do not express NKG2DL on the cell surface. Instead, expression of these ligands is induced upon exposure to cellular stress (e.g., oncogenic transformation or infectious stimuli) to trigger elimination of damaged cells via lysis through NKG2D-receptor-expressing immune cells such as natural killer (NK) cells. Interestingly, NKG2DL surface expression is selectively suppressed in LSC subpopulations, allowing these cells to evade NKG2D-mediated immune surveillance. Here, we present a side-by-side analysis of two different flow cytometry methods that allow the investigation of NKG2DL surface expression on cancer cells i.e., a method involving pan-ligand recognition and a method involving staining with multiple antibodies against single ligands. These methods can be used to separate viable NKG2DL negative cellular subpopulations with putative cancer stem cell properties from NKG2DL positive non-LSC.

We present two different staining protocols for NKG2D ligand (NKG2DL) detection in human primary acute myeloid leukemia (AML) samples. The first approach is based on a fusion protein, able to recognize all known and potentially yet unknown ligands, while the second protocol relies on the addition of multiple anti-NKG2DL antibodies.

NKG2D 利甘德表面检测的两种流细胞学方法，以区分干细胞与急性骨髓性白血病中的散装亚群

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties ...