这项工作得到部分支持, 由科氏研究所支助 (核心) 赠款 P30-CA 14051 从国家癌症研究所, 由国家卫生补助研究所 (CA206157-22 和 GM118066) 和生硬的大理石癌症研究基金安洁莉卡阿蒙。安洁莉卡还是霍华德休斯医学研究所和格伦生物医学研究基金会的研究员。在美国意大利癌症基金会 (AICF) 的支持下, 由玛丽居里行动和意大利癌症研究协会 (AIRC) 的癌症研究奖学金, 以及五年期癌症研究奖学金。他得到了霍华德休斯医学研究所的一份奖学金的支持。

文化条件
RPE-1 hTERT 细胞系培养 Dulbecco 的改良鹰培养基 (材料表) 补充10% 胎牛血清, 2 毫米 l-谷氨酰胺, 100 U/毫升青霉素/链霉素。细胞在37°c 孵化, 5% CO2在一个湿润的环境中。下面描述的协议是使用10厘米的菜肴开发的, 所有的技术细节都是指这些菜肴。如果使用不同的菜肴, 相应地向上或向下缩放。
1. RPE-1 细胞同步
<ol><li>使用新鲜解冻的细胞, 没有经历超过15通道的所有实验。在1天, 解冻和免除 ~ 2.5-5.0 x 105 RPE-1 hTERT 细胞成10厘米的组织培养皿。添加8毫升标准生长培养基和孵化过夜。使用标准单元格计数器 (如 hemocytometer) 确定单元格编号。 注: 细胞密度是指 RPE-1 细胞的生长条件。生长条件可能在不同的细胞系之间变化;考虑相应地调整它们。</li>
	<li>在2天, 通过吸定期生长培养基和增加8毫升培养基, 其中含有5毫米胸苷, 同步 G1/S 边界的细胞。 注: 胸苷浓度和治疗长度可能不同的细胞线。最好进行试验, 以确定最佳条件的细胞线 (s) 进行测试。</li>
	<li>在3天, 24 小时后添加胸苷培养基 (步骤 1.2), 释放从胸苷块的吸气培养基和洗涤细胞三倍的 1x PBS。添加8毫升的定期生长培养基, 并返回板到孵化器。</li>
</ol>2. 通过干扰有丝分裂激酶 Mps1 的活性产生异倍体细胞
<ol><li>在3天, 6 小时后, 从胸苷块释放 (步骤 1.3; 这是约 3-6 小时前细胞进入有丝分裂), 吸入培养基, 一次性冲洗 1x PBS, 并替换为中含有 500 nM reversine (Mps1 抑制剂)。 注: RPE-1 细胞进入有丝分裂 9-12 小时后, 胸苷洗出10,11。然而, 细胞周期的动力学变化在不同的细胞系之间。因此, 通过建立的方法进行细胞周期分析 (例如), 通过测量 DNA 含量通过流动力学分析) 来确定这些动力学在先导实验中是至关重要的。此外, Mps1 抑制剂的最佳工作浓度可能会因细胞系而异。以前使用 RPE1 细胞的实验成功地使用了 reversine 的工作浓度为 500 nM10,11,12。最好进行试验, 以确定最佳浓度的细胞线进行测试。</li>
	<li>在4天, 12 小时后 reversine 治疗 (约6小时后细胞有丝分裂), 吸入 reversine 培养基和洗涤细胞三倍的 1x PBS。将8毫升的定期生长培养基添加到板上, 并将细胞恢复到孵化器中。</li>
</ol>3. 异倍体循环细胞的去除和 ArCK 种群的富集
<ol><li>在6天, 细胞进入第一次有丝分裂后约72小时 (步骤 2.2; 这是大约 66 h 在 reversine 洗涤以后, 对应于 2-3 细胞周期), 抽吸媒介, 洗涤一次与 1x PBS 并且替换与包含300毫微米 nocodazole 的媒介。 注: 最近的工作表明, 窝藏异倍体核型的 RPE-1 细胞 genomically 不稳定, 并在第一次错误有丝分裂10 后, 在 2-3 细胞周期内拘捕其增殖。然而, 这种动力学可能会在不同的细胞线之间变化。因此, 进行实验以确定异倍体循环细胞清除的适当时机是至关重要的。</li>
	<li>在7天, 12 小时后 nocodazole 治疗, 吸入介质, 并添加3毫升 1x PBS 的板。通过敲击盘子的侧面, 摆脱有丝分裂细胞。在每个水龙头之间旋转盘子, 以允许甚至切除有丝分裂细胞。</li>
	<li>为确保完全去除有丝分裂细胞, 重复进行多轮的抖动过程 (建议 3-5 次), 在每轮之间吸出和增加3毫升 1x PBS。</li>
	<li>摇动后, 轻轻地吸入附着在盖子或盘子边缘的液体。简要地, 用10X 放大倍数检查显微镜下的板。确保少数有丝分裂细胞被观察到。如果在10X 目标下发现五多个有丝分裂细胞, 重复另一轮的震荡。 注意: 有丝分裂细胞会出现圆形, 并可能在摇晃后部分分离。在将细胞暴露到下一轮 nocodazole 治疗之前, 确保完全清除有丝分裂细胞是至关重要的。如果不这样做, 可能导致有丝分裂细胞死亡和/或导致二、四倍体杂交细胞的产生。</li>
	<li>最后一次摇动后, 用5毫升的 1x PBS 洗涤细胞。将含有 330 nM nocodazole 的8毫升培养基加入板中, 孵育12小时。</li>
	<li>重复 nocodazole 治疗/抖动 (步骤 3.1-3.5) 每12小时, 直到没有发现有丝分裂细胞后, 孵化。通常, 建议4-5 轮治疗/震动。 注意: 这里提供的信息是指 RPE-1 细胞。不同细胞系间的治疗/震荡次数可能有所不同。为了避免长时间和不必要的暴露于 nocodazole, 在试点试验中, 必须仔细确定有效和彻底清除异倍体循环细胞所需的子弹数量。</li>
	<li>经过 4-5 轮的 nocodazole 治疗/抖动 (步骤 3.1-3.6), 增加10毫升的定期生长培养基和孵化。允许细胞在未来的操作或化验使用前休息 12-24 小时。 注: 在最终的震荡后, 板上剩余的细胞是被逮捕的细胞周期, 如最近的10所示, 港湾复杂的核型。这些细胞被称为 ArCK (用复杂的核型) 细胞被逮捕。在隔离的一周内对 ArCK 的人群进行化验是可取的。</li>
	<li>或者, 要评估被拘捕的细胞与复杂的核型的百分比, 收集和计数的细胞从每次抖动使用标准的细胞计数器。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57215/57215fig1.jpg"> 图 1: 用于生成和隔离具有复杂核型 (ArCK) 和代表性图像的异倍体单元格的方法的总体示意图。(A) ArCK 单元生成和隔离协议示意图。(B)在治疗的每个阶段 RPE-1 hTERT 细胞的代表性图像。最后一个面板 (用红色表示) 代表了独立的 ArCK 单元格。缩放栏100µm.<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57215/57215fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
4. ArCK 人口的特征
<ol><li>通过执行以下化验, 确认成功地隔离 ArCK 人口。<ol>
<li>驴β-乳糖酶染色 (衰老的标志) 使用一个商业上可用的套件 (见材料表)。</li>
		<li>通过使用商业上可用的工具包 (见材料表) 测量促炎性细胞因子 (如 CCL2) 的分泌。</li>
		<li>确认 p53, p21, p16 的细胞周期抑制剂的增加水平。 注意: 为了正确控制这些化验, ArCK 的人群应与 euploid 和异倍体循环细胞进行比较。前者为低通道, 野生型 RPE-1 hTERT 细胞呈指数增长。后者可通过诱导 RPE-1 hTERT 中的染色体错误分离和在第一次异常有丝分裂后收获细胞72小时获得。为此, 请按照上文所述的步骤1.1 中的协议进行, 并在第一个 nocodazole 处理之前收获细胞 (步骤 3.1)。</li>
	</ol>
</li>
	<li>执行单细胞测序, 以评估被拘捕细胞的核型。 注意: 单细胞测序是确定单个细胞级别13上的染色体和亚染色体变化的一种成熟的方法。有关如何执行单单元排序的详细过程, 请在10的其他位置找到。</li>
</ol>

<ol><li>Santaguida, S., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Short-+and+long-term+effects+of+chromosome+mis-segregation+and+aneuploidy%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">Short- and long-term effects of chromosome mis-segregation and aneuploidy.</a> Nature Reviews Molecular Cell Biology. 16 (8), 473-485 (2015).</li>
<li>Pfau, S. J., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromosomal+instability+and+aneuploidy+in+cancer%3A+from+yeast+to+man%5BTitle%5D)+AND+%22EMBO+reports%22%5BJournal%5D)">Chromosomal instability and aneuploidy in cancer: from yeast to man.</a> EMBO reports. 13 (6), 515-527 (2012).</li>
<li>Gordon, D. J., Resio, B., Pellman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Causes+and+consequences+of+aneuploidy+in+cancer%5BTitle%5D)+AND+%22Nature+Reviews+Genetics%22%5BJournal%5D)">Causes and consequences of aneuploidy in cancer.</a> Nature Reviews Genetics. 13 (3), 189-203 (2012).</li>
<li>Holland, A. J., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Boveri+revisited%3A+chromosomal+instability%2C+aneuploidy+and+tumorigenesis%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">Boveri revisited: chromosomal instability, aneuploidy and tumorigenesis.</a> Nature Reviews Molecular Cell Biology. 10 (7), 478-487 (2009).</li>
<li>Meena, J. K., Cerutti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Telomerase+abrogates+aneuploidy-induced+telomere+replication+stress%2C+senescence+and+cell+depletion%5BTitle%5D)+AND+%22The+EMBO+Journal%22%5BJournal%5D)">Telomerase abrogates aneuploidy-induced telomere replication stress, senescence and cell depletion.</a> The EMBO Journal. , 1-14 (2015).</li>
<li>Ohashi, A., Ohori, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aneuploidy+generates+proteotoxic+stress+and+DNA+damage+concurrently+with+p53-mediated+post-mitotic+apoptosis+in+SAC-impaired+cells%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Aneuploidy generates proteotoxic stress and DNA damage concurrently with p53-mediated post-mitotic apoptosis in SAC-impaired cells.</a> Nature Communications. 6, 7668(2015).</li>
<li>Passerini, V., Ozeri-Galai, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+presence+of+extra+chromosomes+leads+to+genomic+instability%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">The presence of extra chromosomes leads to genomic instability.</a> Nature Communications. 7, 10754(2016).</li>
<li>Sheltzer, J. M., Blank, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aneuploidy+drives+genomic+instability+in+yeast%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Aneuploidy drives genomic instability in yeast.</a> Science. 333 (6045), 1026-1030 (2011).</li>
<li>Zhu, J., Pavelka, N., Bradford, W. D., Rancati, G., Li, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Karyotypic+determinants+of+chromosome+instability+in+aneuploid+budding+yeast%5BTitle%5D)+AND+%22PLoS+Genetics%22%5BJournal%5D)">Karyotypic determinants of chromosome instability in aneuploid budding yeast.</a> PLoS Genetics. 8 (5), e1002719(2012).</li>
<li>Santaguida, S., Richardson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromosome+Mis-segregation+Generates+Cell-Cycle-Arrested+Cells+with+Complex+Karyotypes+that+Are+Eliminated+by+the+Immune+System%5BTitle%5D)+AND+%22Developmental+Cell%22%5BJournal%5D)">Chromosome Mis-segregation Generates Cell-Cycle-Arrested Cells with Complex Karyotypes that Are Eliminated by the Immune System.</a> Developmental Cell. 41 (6), 638.e5-651.e5 (2017).</li>
<li>Santaguida, S., Vasile, E., White, E., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aneuploidy-induced+cellular+stresses+limit+autophagic+degradation%5BTitle%5D)+AND+%22Genes+%26+Development%22%5BJournal%5D)">Aneuploidy-induced cellular stresses limit autophagic degradation.</a> Genes & Development. 29 (19), 2010-2021 (2015).</li>
<li>Santaguida, S., Tighe, A., D'Alise, A. M., Taylor, S. S., Musacchio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dissecting+the+role+of+MPS1+in+chromosome+biorientation+and+the+spindle+checkpoint+through+the+small+molecule+inhibitor+reversine%5BTitle%5D)+AND+%22The+Journal+of+Cell+Biology%22%5BJournal%5D)">Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine.</a> The Journal of Cell Biology. 190 (1), 73-87 (2010).</li>
<li>Gawad, C., Koh, W., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+genome+sequencing%3A+current+state+of+the+science%5BTitle%5D)+AND+%22Nature+Reviews+Genetics%22%5BJournal%5D)">Single-cell genome sequencing: current state of the science.</a> Nature Reviews Genetics. 17 (3), 175-188 (2016).</li>
<li>López-Soto, A., Gonzalez, S., López-Larrea, C., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunosurveillance+of+Malignant+Cells+with+Complex+Karyotypes%5BTitle%5D)+AND+%22Trends+in+cell+biology%22%5BJournal%5D)">Immunosurveillance of Malignant Cells with Complex Karyotypes.</a> Trends in cell biology. , 1-4 (2017).</li>
<li>Watson, E. V., Elledge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aneuploidy+Police+Detect+Chromosomal+Imbalance+Triggering+Immune+Crackdown%21%5BTitle%5D)+AND+%22Trends+in+genetics%3A+TIG%22%5BJournal%5D)">Aneuploidy Police Detect Chromosomal Imbalance Triggering Immune Crackdown!</a> Trends in genetics: TIG. , 1-3 (2017).</li>
</ol>

作者没有什么可透露的。

这种新的方法生成和丰富的被逮捕的细胞与复杂的核型 (ArCK) 允许研究的细胞, 有多个染色体的增益或损失, 并停止分裂。该方法的设置是为了方便快速、可靠地隔离 ArCK 细胞。
这项试验中最关键的一步是严格控制药物治疗的时间线, 最重要的是清除异倍体循环细胞。为了获得最佳结果, nocodazole 治疗和抖动的时机特别重要, 以确保循环细胞从板块中移除, 而它们仍然是圆形和有丝分裂, 防止有丝分裂细胞死亡或滑入 G1 的可能性,可能造成二、四倍体杂交人口。不建议在12小时 nocodazole 孵化时, 抖动的时间偏离两个以上的时间。
未来对 ArCK 细胞的研究有可能有助于更深入地了解复杂的核型对细胞生理学的影响。特别是, 最近的一项研究表明, 免疫系统的细胞能够与 ArCK 细胞的免疫清除功能进行交互并触发10。这里描述的方法为 ArCK 细胞的进一步表征提供了一个良好的出发点, 包括澄清未变形细胞免疫清除的分子机制和致癌转化的研究可以绕过此监视机制, 这是字段14、15中的关键问题。

染色体分离过程中的错误导致体检, 这种情况的特征是染色体数不是单倍体补充的倍数1,2,3,4。异倍体核型触发复制重音产生进一步的基因组不稳定5, 6, 7, 8, 9, 10, 增加核型复杂性, 并最终导致细胞周期逮捕的细胞亚群10。这里提出的方法的目的是生成和分离这样的亚群从循环细胞。通过使用廉价的组织培养技术, 该协议促进了细胞周期被捕获的复杂核型异倍体细胞的分离和鉴定。这些细胞被称为 ArCK (以复杂的核型) 细胞及其 euploid 循环对应物作为对照。
该议定书是第一个为此目的建立, 并允许隔离和进一步研究 ArCK 线包括, 但不限于, 其诱发衰老和衰老相关分泌表型 (SASP), 他们的亲炎特征, 以及它们与免疫细胞接触的能力。这里提出的方法已经在未变形, 永生化的人类细胞, 但尚未在癌症线测试。由于抑制一个或多个通路, 某些转化细胞可能对细胞周期停止敏感;因此, 应在其他单元格行中执行进一步的验证。

<table><tbody><tr><td>DMEM (Dulbecco&amp;rsquo;s Modified Eagle Medium)&amp;nbsp;</td><td>Thermofisher</td><td>11995-073</td><td></td></tr><tr><td>Thymidine</td><td>Sigma Aldrich</td><td>T1859</td><td></td></tr><tr><td>Reversine</td><td>Cayman Chemical Company</td><td>10004412</td><td></td></tr><tr><td>Nocodazole</td><td>Sigma Aldrich</td><td>M1410</td><td></td></tr><tr><td>Anti-p53 antibody</td><td>Santa Cruz</td><td>sc-126</td><td></td></tr><tr><td>Anti-p21 antibody</td><td>Santa Cruz</td><td>sc-6246</td><td></td></tr><tr><td>Anti-p16 antibody</td><td>BD</td><td>554079</td><td></td></tr><tr><td>Senescence b-Galactosidase Staining Kit</td><td>Cell Signaling Technology&amp;nbsp;</td><td>9860</td><td></td></tr><tr><td>Proteome Profiler Human Cytokine Array Kit&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>ARY005B</td><td></td></tr></tbody></table>

generation and isolation of cell cycle-arrested cells with complex karyotypes

染色体错误隔离导致体检, 这种情况下细胞的染色体数目不平衡。几行证据有力地表明, 体检引发基因组不稳定, 最终产生具有复杂核型的细胞, 从而阻止其增殖。对具有复杂核型的细胞进行分离和表征, 对于研究染色体数目不平衡对细胞生理学的影响至关重要。迄今为止, 还没有建立可靠隔离这种异倍体细胞的方法。本文提供了一种利用标准的、廉价的组织培养技术对复杂核型异倍体细胞进行富集和分析的协议。该协议可用于分析具有复杂核型的异倍体细胞的几种特征, 包括其诱导的衰老相关的分泌形态、炎症性质和与免疫细胞相互作用的能力。因为癌细胞常常会造成染色体数量的失衡, 所以破译体检如何影响正常细胞的细胞生理学是至关重要的, 最终目的是揭露其亲和抗癌变的作用。

这种方法利用一个体外组织培养系统来隔离异倍体细胞, 其复杂的核型可以阻止其增殖。这个种群被称为 ArCK (以复杂的核型) 细胞被拘捕。图 1A显示了实验方案。野生型循环 RPE-1 hTERT 细胞在 G1/S 边界与胸苷同步, 并与 Mps1 抑制剂进行治疗, 以诱导染色体错误分离。在诱导染色体错误隔离后, 将主轴毒物 nocodazole 添加到细胞中72小时。12小时 nocodazole 治疗后, 仍能增殖的异倍体细胞进入有丝分裂, 并由于干扰微管聚合而被困在这个细胞周期阶段。这些被困的细胞, 然后很容易删除的温和的震动。这一震动过程重复了 3-5 次, 以确保完全清除循环细胞。图 1B显示了在治疗的每个阶段 RPE-1 细胞的代表性图像。
与 euploid 和异倍体循环单元相比, ArCK 细胞显示的细胞周期抑制剂 (如 p53、p21 和 p16) 的水平增加,如图2A 所示。此外,图 2B显示 ArCK 细胞中的正β-乳糖酶染色, 与 euploid 控制细胞相比, 它是细胞衰老的一个成熟标记。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57215/57215fig2.jpg"> 图 2: 使用 ArCK 单元格的代表性结果.(A)在 euploid、异倍体循环和 ArCK 细胞中评估细胞周期抑制剂水平的西方印迹。p53、p21 和 p16 的含量由西方印迹分析确定。肌动蛋白作为装载控制。(B) β-乳糖酶染色法评估 ArCK 细胞的衰老程度。在 euploid 细胞和 ArCK 细胞中测定了衰老相关的β-乳糖酶 (β-加仑) 活性。缩放栏100µm.<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57215/57215fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
这种细胞周期骤停是异倍体细胞具有复杂核型的显著特征, 如从图3中的单细胞测序中的代表性染色体分析显示, 其中 ArCK 细胞显示多个随机染色体增益和损失。此发现与以前的报表10完全一致。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57215/57215fig3.jpg"> 图 3: ArCK 单元格的代表性单细胞测序.显示六个代表性 ArCK 细胞的核型的分割图。分割图在日志2尺度上显示所有染色体从1到 X 的复制号相对于 euploid 引用 (野生类型 RPE-1 是雌性原点的双倍细胞线)。染色体的增加是红色的, 染色体的损失在绿色突出显示。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57215/57215fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes at JoVE.com

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

体检导致基因组不稳定, 最终产生细胞周期-被捕获的细胞与复杂的核型。本文提供了一种简单方便的分离异倍体细胞的方法。

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 ...

generation-isolation-cell-cycle-arrested-cells-with-complex

Research

JoVE Journal

Cancer Research

10.4K 次观看.  Massachusetts Institute of Technology. 体检导致基因组不稳定, 最终产生细胞周期-被捕获的细胞与复杂的核型。本文提供了一种简单方便的分离异倍体细胞的方法。

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

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

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

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

通过两个互补细胞同步方案研究细胞周期调控的基因表达

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

科研

遗传学

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

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.

This protocol describes the use of centrifugal elutriation to separate primary acute lymphoblastic leukemia cells into different cell cycle phases.

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 ...

癌症研究

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

哺乳动物细胞周期内蛋白质激酶 rna 激活的 rna 相互作用研究

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

生物化学

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

发育生物学

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

为核型分析人类胚胎干细胞的染色体蔓延制备

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

生物学

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

成人和胚胎骨骼的肌肉Microexplant文化和骨骼肌肉干细胞的分离

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Analysis of Cell Cycle Position in Mammalian Cells

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

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

在哺乳动物细胞中细胞周期位置分析

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

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

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

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

代谢标记和流式细胞仪测定细胞周期进程的动力学

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

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 ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

无细胞化验，研究染色质解聚在有丝分裂的结束

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...