Name: preparation of primary acute lymphoblastic leukemia cells in different cell cycle phases by centrifugal elutriation
Uploaded: 2017-11-10T14:00:00.000+00:00
Duration: 9 min 9 s
Description: Watch this Scientific Journal Video about Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation at JoVE.com

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>

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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><tbody><tr><td>Hanks&#039; balanced salt solution</td><td>Lonza</td><td>10-508Q</td><td>1 L bottle</td></tr><tr><td>Fetal Plus bovine serum</td><td>Atlas Biologicals</td><td>FP-0500-A</td><td>500 mL bottle</td></tr><tr><td>2-napthol-6,8-disulfonic acid dipotassium salt</td><td>Acros Organics</td><td>212-672-4</td><td>100 g powder</td></tr><tr><td>50 mL concial tubes</td><td>Denville Scientifics</td><td>C1062-P</td><td>500/case</td></tr><tr><td>PES Membrane 0.22 &amp;micro;m filter unit</td><td>Millipore</td><td>SLGP033RB</td><td>250/pack</td></tr><tr><td>Avanti J-26S XPI w/ Elut. Non-IVD - 50/60 Hz, 200/208/240V</td><td>Beckman Coulter</td><td>B14544</td><td>centrifuge compatible with elutriation system</td></tr><tr><td>JE 5.0 elutriator rotor kit</td><td>Beckman Coulter</td><td>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><td>Chamber, standard, 4-mL, &quot;A&quot;</td><td>Beckman Coulter</td><td>356943</td><td>standard elutriation chamber&amp;nbsp;</td></tr><tr><td>Masterflex L/S Easy-Load pump head</td><td>Cole-Parmer</td><td>EW-07518-10</td><td></td></tr><tr><td>Masterflex L/S Variable-Speed Drive</td><td>Cole-Parmer</td><td>EW-07528-10</td><td></td></tr><tr><td>Masterflex BioPharm platinum-cured silicone pump tubing, L/S 16</td><td>Cole-Parmer</td><td>EW-96420-16</td><td></td></tr><tr><td>25 G x 1.5 Precision Glide needle</td><td>BD</td><td>305127</td><td>sterile, single-use needles</td></tr><tr><td>10 mL Luer-Lok syringe</td><td>BD</td><td>309604</td><td>sterile, single-use syringes</td></tr><tr><td>Vi-Cell XR</td><td>Beckman Coulter</td><td>383556</td><td></td></tr><tr><td>PI/RNase staining buffer</td><td>BD Pharmingen</td><td>550825</td><td>propidium iodide/RNase staining buffer</td></tr><tr><td>cyclin B1 (GNS1) mouse monoclonal IgG antibody</td><td>Santa Cruz Biotechnology</td><td>sc-245</td><td></td></tr><tr><td>cyclin D1 (DCS-6) mouse monoclonal IgG antibody</td><td>Santa Cruz Biotechnology</td><td>sc-20044</td><td></td></tr><tr><td>P-Rb (S807/811) (D20B12) XPR rabbit monoclonal antibody</td><td>Cell Signaling Technology</td><td>8516s</td><td></td></tr><tr><td>GAPDH (14C10) rabbit monoclonal antibody</td><td>Cell Signaling Technology</td><td>2118s</td><td></td></tr><tr><td>Goat anti-mouse IgG (H+L)-HRP conjugate</td><td>BioRad</td><td>170-6516</td><td></td></tr><tr><td>Goat anti-rabbit IgG (H+L)-HRP conjugate</td><td>BioRad</td><td>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

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western countries. The majority of CLL patients show an indolent course of the disease as well as an anergic phenotype of their leukemia cells, referring to a B cell receptor unresponsive to external stimulation. We have recently shown that the transcription factor NFAT2 is a crucial regulator of anergy in CLL. A major challenge in the analysis of the role of a transcription factor in different diseases is the identification of its specific target genes. This is of&#160;great significance for the elucidation of pathogenetic mechanisms and potential therapeutic interventions. Chromatin immunoprecipitation (ChIP) is a classic technique to demonstrate protein-DNA interactions and can, therefore, be used to identify direct target genes of transcription factors in mammalian cells. Here, ChIP was used to identify LCK as a direct target gene of NFAT2 in human CLL cells. DNA and associated proteins are crosslinked using formaldehyde and subsequently sheared by sonication into DNA fragments of approximately 200-500 base pairs (bp). Cross-linked DNA fragments associated with NFAT2 are then selectively immunoprecipitated from cell debris using an &#945;NFAT2 antibody. After purification, associated DNA fragments are detected via quantitative real-time PCR (qRT-PCR). DNA sequences with evident enrichment represent regions of the genome which are targeted by NFAT2 in vivo. Appropriate shearing of the DNA and the selection of the required antibody are particularly crucial for the successful application of this method. This protocol is ideal for the demonstration of direct interactions of NFAT2 with target genes. Its major limitation is the difficulty to employ ChIP in large-scale assays analyzing the target genes of multiple transcription factors in intact organisms.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. NFAT transcription factors are important regulators of development and activation in numerous cell types. Here, we present a protocol for the use of chromatin immunoprecipitation (ChIP) in human CLL cells to identify novel target genes of NFAT2.

一种检测慢性淋巴细胞白血病新 nfat2 靶基因的染色质免疫沉淀法

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

科研

癌症研究

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 ...

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

从2DE凝胶点在蛋白质功能:在课慢性淋巴细胞白血病学到HS1

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

医学

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

一个简单的红细胞溶解法对B淋巴母细胞系的建立

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

免疫与感染

Modeling Chemotherapy Resistant Leukemia In Vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.

建模化疗耐药白血病<em&gt;体外</em

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and ...

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

分离的CD133 +肝干细胞的克隆扩增

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

生物学

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

染色体制备培养细胞

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

Preparation of Subcellular Fractions from Leukemic Cells: A Method to Generate Cellular and Nuclear Fractions from Leukemic Cells

Source: Hay, J., et al. <a href="https://www.jove.com/t/60426">Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking.</a> J. Vis. Exp. (2019).
This video describes the preparation of subcellular fractions from leukemic cells which is a method to generate cellular and nuclear fractions from leukemic cells. The nuclear and cytoplasmic fractions can be used for further downstream analysis.

从白血病细胞制备亚细胞组分:从白血病细胞生成细胞和细胞核组分的方法

Source: Hay, J., et al. Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking. J. ...

JoVE Encyclopedia of Experiments

白血病

In vitro techniques & assays

Density Gradient Centrifugation: A Method to Isolate CLL Cells from Peripheral Blood

Source: Hay, J. et al. <a href="https://www.jove.com/t/60426">Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking.</a> J. Vis. Exp. (2019).
This video describes the technique of isolating chronic lymphocytic leukemia (CLL) cells from peripheral blood. With this protocol, we will isolate CLL cells from human patient blood sample for subsequent efficient generation of nuclear and cytoplasmic fractions.

密度梯度离心:一种从外周血中分离 CLL 细胞的方法

Source: Hay, J. et al. Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking. J. Vis. ...

Cell isolation & characterization

Leukemic Subpopulation Harvest: A Method for Spatial Separation of Leukemic Cell Subpopulations from 2D Co-culture

Source: Slone, W. L., et al. <a href="https://www.jove.com/t/53645">Modeling Chemotherapy Resistant Leukemia In Vitro.</a> J. Vis. Exp. (2016).
This video describes a method for spatial separation of three leukemic cell subpopulations: the suspended leukemic cells freely floating in the media, phase bright leukemic cells adhered to the surface of the mesenchymal stromal cell monolayer, and the phase dim leukemic cells that have migrated beneath the mesenchymal stromal cell monolayer from 2D co-culture.

白血病亚群收获:一种从 2D 共培养中空间分离白血病细胞亚群的方法

Source: Slone, W. L., et al. Modeling Chemotherapy Resistant Leukemia In Vitro. J. Vis. Exp. (2016).
This video describes a method for spatial separation ...