我们感谢Zubiaga和Altmeyer实验室的成员进行有益的讨论和技术支持。这项工作得到了西班牙部（SAF2015-67562-R，MINECO / FEDER，UE），巴斯克政府（IT634-13和KK-2015/89）和巴斯克历史大学UPV / EHU（ UFI11 / 20）。

细胞周期进展的细胞同步，释放和监测<ol><li> 基于胸腺嘧啶和诺考达唑（Thy-Noc）的同步和释放的有丝分裂的U2OS细胞 <ol><li>准备所需的细胞培养基。 U2OS细胞通常在补充有10％（体积/体积）FBS（任选的:1％青霉素/链霉素）的DMEM-谷氨酰胺培养基中生长。在无菌条件下进行所有培养基制备和操作，并在使用前将补充培养基（从现在称为"完全培养基"）升温至37℃。 </li><li>在10毫升完全培养基中每100毫升培养皿中种子2×10 6个 U2OS细胞。为了计算所需的培养皿数量，考虑到每个100毫米的培养皿通常提供足够的有丝分裂细胞来重新铺板6孔板的大约5个孔（0.2-0.25×10 6个细胞/孔）（参见图1B ）。每个选定时间点需要两口井在实验中（1孔用于RNA提取，1孔用于细胞周期监测）。另外，可以每个时间点收集第三个孔用于蛋白质分析。 注意:晚上（晚上7点左右）的平​​板细胞，以便随后的步骤可以在后几天的工作时间内进行。包括2个异步生长细胞的孔以定义FACS分析补偿设置。 </li><li>通过在37℃下在5％CO 2的湿润气氛中孵育100毫米培养皿24小时使细胞附着。 </li><li>对于胸苷块，通过将145.2mg胸苷粉末溶解在3mL H 2 O（或等量的量）中制备200mM胸苷储液，并通过0.2μm孔径过滤器过滤灭菌溶液。稍微加温可能有助于溶解胸苷。将100μL新鲜制备的200mM储备液加入到每个100mm培养皿（终浓度2mM）中。 37℃孵育细胞与胸苷20小时76; C在具有5％CO 2的加湿气氛中。 注意:晚上（晚上7点左右）处理细胞，第二天有时间进行胸苷释放和诺考达唑阻滞。 </li><li>要从胸苷块中释放，请在第二天（3点）下午清除含胸苷生长培养基。用预热的1×PBS洗涤细胞两次，并向每个100mm的培养皿中加入10mL的完全培养基。在含5％CO 2的潮湿气氛中，37℃孵育细胞5小时。 </li><li>对于有丝分裂细胞停滞，加入诺考达唑至终浓度为50 ng / mL（8 pm）。通过将诺考达唑粉末溶于DMSO（ 如 5 mg / mL）中制备储液，并在-20°C下冷冻保存。在含5％CO 2的潮湿气氛中，37℃下，使用诺考达唑孵育不超过10-11小时的细胞。 </li><li>在早期M期（有丝分裂脱落）中释放诺考达唑介导的停滞，并在几个时间点收集样品nts（从6-7 am开始）。 <ol><li>通过摇动每个平板并轻轻吸取含有含诺科达唑的生长培养基，分离圆形（有丝分裂）细胞。将分离的细胞从每个100mm平板收集到50mL无菌管中，离心（300×g，5分钟，室温（RT）），并通过加入1x PBS洗涤细胞两次，然后离心。建议使用冷PBS或PBS加诺考达唑以避免有丝分裂滑脱（参见讨论部分）。 </li><li>将每个100mm平板上收集的有丝分裂细胞重悬于10 mL完全培养基中。保存2 mL RNA提取，2 mL进行FACS分析，时间为0 h（每个样品约0.2-0.25 x 10 6个细胞）。 </li><li>在6孔板（2mL /孔; 0.2-0.25×10 6个细胞/孔）中重新平板剩余的有丝分裂细胞用于随后的时间点。 注意:请记住，每个选定时间点需要2个孔（RNA为1个，FACS分析为1个）。 </li></ol></li><li>收集样品有时间点建议每1.5至3小时以获得足够的细胞周期进展曲线。 <ol><li>对于RNA提取，取出培养基，用2 mL预温的1x PBS冲洗干净，并在孔中加入1 mL合适的RNA分离试剂（ 如 TRIzol）（在化学品安全柜中执行最后一步）。上下移动以分离和裂解细胞，将细胞裂解液转移至1.5 mL微量离心管，在室温下孵育5分钟，并将试管储存在-80°C直至进一步使用。 </li><li>对于FACS分析，用2mL预温的1x PBS冲洗，加入预先加热的胰蛋白酶-EDTA溶液（0.3mL /孔）以分离细胞，通过加入1mL完全培养基封闭胰蛋白酶-EDTA，并将每个样品分开收集15 mL管。 <ol><li>离心细胞（300×g，5分钟，RT），保存细胞沉淀并弃去上清液。为了修复细胞，通过轻轻涡旋管将1ml冷冻的70％（v / v）乙醇的1x PBS中的细胞重新悬浮，并将其放置在冰上，供应用在4℃下储存约15分钟或通过FACS进行染色进一步分析（在步骤1.4-1.5中描述）。 </li></ol></li></ol></li></ol></li><li> 基于HU的同步和从G1 / S边界释放U2OS细胞 <ol><li>如步骤1.1所述准备完整的细胞培养基。 </li><li>在6孔板（每孔2mL完全培养基）中每孔种0.25×10 6个 U2OS细胞。为了计算实验所需的孔数，考虑到每个选定时间点需要2个孔（RNA提取1个孔，细胞周期监测1个孔），另外2个异步生长细胞的孔是需要定义FACS分析补偿设置。 </li><li>通过在37℃下在5％CO 2的潮湿气氛中孵育6孔板过夜（O / N）使细胞附着。 </li><li>第二天早上从井中取出完整培养基，加入2 mL的预热的无FBS的DMEM-谷氨酰胺培养基。在37℃，5％CO 2的潮湿气氛中孵育细胞24小时。 注意:在所有孔中执行此步骤，除了2（保存定义FACS设置）。如果通过简单地将细胞与HU孵育来实现有效的同步，则可以省略血清提取步骤。 </li><li>用HU抑制G1 / S细胞周期。 <ol><li>每次使用前准备新鲜的HU储备溶液（500 mM）。加入2mL H 2 O至76.06mg HU粉末，混匀至充分溶解。通过0.2μm孔径过滤器过滤灭菌溶液。将50 mL完全培养基与400μL过滤灭菌的HU储备液混合，最终HU浓度为4 mM。 </li><li>除了从定义FACS设置所需的2个孔以外的所有孔中取出培养基，并用新鲜制备的4mM含HU的完全培养基（2mL /孔）替换。 </li><li>孵育细胞24小时含HU的培养基在37℃，5％CO 2的加湿气氛中。 </li></ol></li><li>从HU介导的逮捕中释放细胞。从孔中取出含HU的培养基，并用预热的1×PBS（每次2mL）冲洗两次。每孔加入2 mL完全培养基。收集0个时间点的2个样品（1个用于RNA提取，1个用于FACS进行细胞周期停滞验证），以及2个样品保存用于FACS设置。将剩余的孔放在培养箱中。 </li><li>每1.5至3小时收集样品，以获得足够的细胞周期进程分布。在每个时间点，进行1.1.8.1-1.1.8.2中所述的样品处理（RNA提取和FACS分析）。 </li></ol></li><li> 用DNA损伤剂治疗 注意:每当目的是澄清化合物（ 例如 DNA损伤剂）对细胞周期事件的影响时，任何前述的同步方法可以是结合用基因毒素治疗细胞。为了选择同步方法，重要的是考虑我们想要分析的细胞周期的相位。通常，Thy-Noc程序可能适合于研究化合物在G1期或S期进入中的作用，而HU介导的同步可能更适合研究S至G2期或有丝分裂期的影响。 <ol><li>分析基因毒素在G1期或S期进入中的作用<ol><li>按照1.1.2所述同步单元格。至1.1.6。 </li><li>从诺考达唑释放细胞，并按照1.1.7所述重新平板化。在37℃下，在5％CO 2的潮湿气氛中孵育3小时，以便在添加剂之前将其连接（所需的孵育期可能因细胞系而异）。 </li><li>添加代理并收集样品，如1.1.8中所述。 </li></ol></li><li>基因毒素在SG中的作用分析2阶段或M进入<ol><li>按1.2.2所述同步单元格。至1.2.5。 </li><li>如1.2.6所述从HU释放细胞。并直接添加代理。 </li><li>收集样品，如前文1.8所述。 </li></ol></li></ol></li><li> 通过碘化丙啶（PI）染色和FACS分析监测细胞同步和通过细胞周期的进展 注意:所有时间点收集的样品以及定义FACS设置所需的样品一旦固定就可以在4°C储存（如1.1.8.2中所述）。用PI溶液进行染色，然后同时对所有实验样品进行FACS分析。当在535nm激发，宽600nm左右的发光峰时，PI插入到双链DNA的主槽中，产生高度荧光信号。由于PI还可以结合双链RNA，因此需要用RNase处理细胞以获得最佳的DNA分辨率。 <ol><li>准备新鲜的PI染色溶液。可以通过将PI粉末溶解在PBS（ 例如 5mg / mL）中来制备PI储备溶液。储存溶液在4°C（黑暗）。染色溶液由PI（140μM），柠檬酸钠（38mM）和Triton X-100（0.01％v / v）组成。 </li><li>将适当的表面（ 例如烤箱）预热至37°C。 </li><li>离心固定细胞（450 x g，5 min，RT），倾析上清液（乙醇），并用1x PBS洗涤一次。 </li><li>再次离心细胞，除去PBS，每样品加入300μLPI染色溶液（除了其中一个样品用于FACS设置;将PBS添加到该样品中）。 </li><li>将细胞转移至FACS管（5mL圆底聚苯乙烯管）。 </li><li>向每个样品加入1μLRNA酶A，混合并在37°C的黑暗条件下孵育样品30分钟。样品可以保存在4°C的光照条件下，最长不得超过2-3天。 </li><li>通过流程分析样品中的DNA含量流式细胞仪。使用PI染色的异步样本定义FACS分析补偿设置。使用空白样品（不含PI染色溶液）检查自体荧光。以前已经描述了PI染色介导的通过流式细胞术分析DNA含量的基础。 </li></ol></li><li> 通过双磷酸H3（Ser 10）/ PI染色测定有丝分裂指数 注意:通过流式细胞术可以轻易地检测到丝氨酸10（pH3）中对磷酸化组蛋白H3特异性抗体的细胞进行有丝分裂。伴随的PI染色可用于确定细胞群体基于DNA含量的分布。 FACS设置的最佳配置需要5个样品:空白，仅限PI，仅限pH3，仅次要抗体和双重染色。 <ol><li>离心固定细胞（450×g，5分钟，4℃）并弃去上清液。描述以下步骤以在15mL管中进行染色。 </li><li>通过加入1洗涤细胞mL PBS-T（PBS + 0.05％Tween-20）沉淀并离心（450×g，5分钟，4℃）。去除上清液。 </li><li>在100-200μLPBS-T + 3％BSA中加入稀释（1:500）的抗pH3抗体，并在RT（或O / N在4℃）下摇摆培养2小时。 </li><li>加入2mL PBS-T（PBS + 0.05％Tween-20）并离心（450×g，5分钟，4℃）。去除上清液。 </li><li>再通过向沉淀中加入2mL PBS-T再次洗涤，离心并弃去上清液。 </li><li>在100-200μL的PBS-T + 3％BSA中稀释（1:500）二次抗体（在选择的pH3抗体的情况下为抗兔AlexaFluor 488），并在室温下摇摆1小时（或O / N在4℃）。保护样品免受光照。 </li><li>通过如步骤1.5.4所述的离心，用PBS-T（2mL）洗涤两次。 </li><li>按照所述进行PI染色（步骤1.4.1至1.4.6）。 </li></ol></li></ol> 2.基因表达分析的样本收集和处理<ol><li>采取将RNA分离试剂中的1.5 mL微量离心试样从冷冻箱中取出，并让其在室内解冻，并在化学品安全柜内解冻。 </li><li>向每个样品中加入400μL氯仿，并剧烈振荡（但不要旋涡）直到完全混合。在室温下孵育样品5分钟。 </li><li>在台式微量离心机中离心管15分钟（≥8,000xg，4°C）。 </li><li>将水相（上）相转移到新的1.5 mL微量离心管中，并记录转移的体积（为了简化程序，建议在实验的所有样品中收集相等的体积）。 </li><li>在混合的同时，向水相中逐滴加入1体积％的100％乙醇。不要离心</li><li>使用商业RNA mini准备工具执行下一步。将每个样品多达700μL，包括可能形成的任何沉淀物放入2 mL收集管（制造商提供）的旋转柱中。 </li><li>关上盖子和离心机（≥8,000g，RT）15 s。丢弃流通。重复上一步与剩余的样品（如果有的话）。 </li><li>按照制造商的说明进行RNA洗涤和洗脱（在30-40μl不含核酸酶的H 2 O中洗脱每个样品，以获得适合的下一步RNA浓度）。 </li><li>通过吸光度测量确定样品的RNA浓度和纯度（ A 260/280比为2.0-2.1表示RNA样品的良好纯度）。将RNA样品储存于-80°C直到用于RT-qPCR分析。 </li><li>对于RNA转化成cDNA和随后的定量PCR，每个样品取1μgRNA，并根据制造商的说明书制备逆转录酶反应。获得的cDNA样品可以在4℃（几天）或-20℃下保存（更长时间）。 注意:实时PCR的样品制备，引物设计和其他注意事项已经例如在文献22,23中有所描述。 </li></ol>

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作者没有什么可以披露的。

在细胞周期中涉及短暂和特异性作用的微调调节基因的分析需要均匀的细胞群体。许多研究人员通常使用长期建立的肿瘤细胞系用于这些目的，并且已经开发了多种方法以获得同步（或部分同步的）细胞群体，目的是在确定的细胞周期阶段中积累尽可能多的细胞。此外，已经作出了大量努力，以改进和优化建立良好的同步方式。然而，所有同步协议都有缺点，这可能归因于细胞培养物的异质性，?...

通过细胞周期的所有阶段的过渡与紧密调节的基因表达程序相结合。认为在整个细胞周期中协调基因转录的"开"和"关"是在复杂的转录调节系统的控制之下，不仅调节时间，而且调节基因表达的水平。已知解除关键细胞周期成分的调节有助于几种疾病的发展，并且是肿瘤发生的已知标志1,2 。在酵母和哺乳动物细胞中进行的全基因组转录组学分析显示，大量基因在细胞周期中表现出周期性的基因表达模式，表明细胞周期内的转录波动反映了给定基因产物的时间需求在精确的阶段3，4 ， </sup&gt; 5 。 研究细胞周期调节基因表达的主要任务是将细胞同步到特定的细胞周期阶段。细胞同步有助于解释基因表达模式与特定细胞周期相变的关联，并且导致更好地了解许多基因的调节和功能。细胞同步对于研究抗癌药物的作用机制也是重要的，因为已知化学治疗剂影响基因表达以及细胞周期动力学6,7 。然而，通常难以确定由这些药剂治疗引起的基因表达差异是否是对治疗的直接反应，或者仅仅是细胞周期特征变化的结果。为了区分这些可能性，基因表达应该在已经存在的细胞中进行分析在加入化疗药物之前同步化。 除了一些原代细胞例如新鲜分离的淋巴样细胞，其构成在G0-8中同步的均质细胞群体， 体外建立的细胞系在培养物中异生生长。在常规生长条件下，这些异步循环细胞发现于细胞周期的所有阶段，但优先在G1 9中 。因此，本文不提供特定细胞周期阶段功能或基因表达分析的最佳方案（ 如 G1，S等）。未转化的永生化细胞系（ 例如成纤维细胞）可以与所谓的生理方法10同步。这些方法基于未转化细胞的保留的原代细胞特征，例如细胞接触抑制和生长因子依赖性，以便继续循环。切除的血清与接触抑制组合使得非转化细胞在G0 / G1处停滞。然而，同步的细胞周期进入和进展通常需要传代培养，这也涉及细胞的人工脱离和再镀覆10 。最重要的是，该方法不适用于转化细胞系的同步，绝大多数目前使用的已建立的细胞系，其特征在于缺乏细胞接触介导的生长抑制或对生长因子撤出的应答。因此，很明显，在细胞周期的特定阶段中需要替代方法用于有效的细胞同步。一般来说，最常用的同步方法是基于细胞周期的一个定义点的瞬时化学或药理学抑制，通常是DNA合成或有丝分裂纺锤体形成。抑制DNA合成通过在晚期G1期或早期S期停滞细胞来同步细胞。这可以是achi通过添加化合物，如拟合物，核苷酸生物合成抑制剂11,12 ， aphidicolin，DNA聚合酶抑制剂13,14 ，羟基脲，核糖核苷酸还原酶15,16抑制剂或过量的胸苷17,18 等化合物。另一方面，微管聚合抑制剂如秋水仙碱或诺考达唑能够阻止有丝分裂纺锤体形成，导致早期M期19,20,21细胞同步。 在这项工作中，我们描述了一种基于瞬时化学抑制的两个互补同步协议的方法，用于研究mRNA上的细胞周期调节基因的表达水平。这种方法是定义细胞周期基因在特定细胞周期过程中的作用的基础。此外，它为研究抗癌治疗的影响提供了一个总体框架，以准确地检测药物反应性基因，并最大限度地减少由这些药物产生的细胞周期进程扰动产生的误解。

<table><tbody><tr><td>DMEM, high glucose, GutaMAX supplement</td><td>Thermo Fisher Scientific</td><td>61965-059</td><td></td></tr><tr><td>FBS, qualified, E.U.-approved, South America origin</td><td>Thermo Fisher Scientific</td><td>10270-106</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Thermo Fisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>0.25% Trypsin-EDTA (1x), phenol red</td><td>Thermo Fisher Scientific</td><td>25200-072</td><td></td></tr><tr><td>Thymidine</td><td>SIGMA</td><td>T1895-5G</td><td>Freshly prepared. Slight warming might help dissolve thymidine.</td></tr><tr><td>Nocodazole</td><td>SIGMA</td><td>M-1404</td><td>Stock solution in DMSO stored at -20 &amp;ordm;C in small aliquots</td></tr><tr><td>Hydroxyurea</td><td>SIGMA</td><td>H8627</td><td>Freshly prepared</td></tr><tr><td>Mitomycin C from Streptomyces caespitosus</td><td>SIGMA</td><td>M4287</td><td>1.5 mM stock solution in sterile H&lt;sub&gt;2&lt;/sub&gt;O protected from light and stored at 4 &amp;ordm;C</td></tr><tr><td>Dimethyl sulfoxide</td><td>SIGMA</td><td>D2650</td><td></td></tr><tr><td>Propidium iodide</td><td>SIGMA</td><td>P4170</td><td>Stock solution in sterile PBS at 5 mg/ml, stored at 4 &amp;ordm; C protected from light.</td></tr><tr><td>PBS pH 7.6</td><td></td><td></td><td>Home made</td></tr><tr><td>Ethanol</td><td>PANREAC</td><td>A3678,2500</td><td></td></tr><tr><td>Chloroform</td><td>SIGMA</td><td>C2432</td><td></td></tr><tr><td>Sodium Citrate</td><td>PANREAC</td><td>131655</td><td></td></tr><tr><td>Triton X-100</td><td>SIGMA</td><td>T8787</td><td></td></tr><tr><td>RNAse A</td><td>Thermo Fisher Scientific</td><td>EN0531</td><td></td></tr><tr><td>TRIzol Reagent</td><td>LifeTechnologies</td><td>15596018</td><td></td></tr><tr><td>RNeasy Mini kit</td><td>QIAGEN</td><td>74106</td><td></td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit</td><td>Thermo Fisher Scientific</td><td>4368814</td><td></td></tr><tr><td>Anti-Cyclin E1 antibody</td><td>Cell Signaling</td><td>4129</td><td>1:1000 dilution in 5% milk, o/n, 4 &amp;ordm;C</td></tr><tr><td>Anti-Cyclin B1 antibody</td><td>Cell Signaling</td><td>4135</td><td>1:1000 dilution in 5% milk, o/n, 4 &amp;ordm;C</td></tr><tr><td>Anti-&amp;beta;-actin</td><td>SIGMA</td><td>A-5441</td><td>1:3000 dilution in 5 % milk, 1 hr, RT</td></tr><tr><td>Anti-pH3 (Ser 10) antiboty</td><td>Millipore</td><td>06-570</td><td>Specified in the protocol</td></tr><tr><td>Secondary anti-rabbit AlexaFluor 488 antibody</td><td>Invitrogen</td><td>R37116</td><td>Specified in the protocol</td></tr><tr><td>Secondary anti-mouse-HRP antibody</td><td>Santa Cruz Biotechnology</td><td>sc-3697</td><td>1:3000 dilution in 5 % milk, 1 hr, RT</td></tr><tr><td>Forward E2F1 antibody (human)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; TGACATCACCAACGTCCTTGA</td><td>Biolegio</td><td></td><td>Designed by PrimerQuest tool (https://eu.idtdna.com/site)</td></tr><tr><td>Reverse E2F1 antibody (human)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; CTGTGCGAGGTCCTGGGTC</td><td>Biolegio</td><td></td><td>Designed by PrimerQuest tool (https://eu.idtdna.com/site)</td></tr><tr><td>Forward E2F7 antibody (human)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; GGAAAGGCAACAGCAAACTCT</td><td>Biolegio</td><td></td><td>Designed by PrimerQuest tool (https://eu.idtdna.com/site)</td></tr><tr><td>Reverse E2F7 antibody (human)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; TGGGAGAGCACCAAGAGTAGAAGA</td><td>Biolegio</td><td></td><td>Designed by PrimerQuest tool (https://eu.idtdna.com/site)</td></tr><tr><td>Forward p21&lt;sup&gt;Cip1 &lt;/sup&gt;antibody (human)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; AGCAGAGGAAGACCATGTGGAC</td><td>Biolegio</td><td></td><td>Designed by PrimerQuest tool (https://eu.idtdna.com/site)</td></tr><tr><td>Reverse p21&lt;sup&gt;Cip1 &lt;/sup&gt;antibody (human)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; TTTCGACCCTGAGAGTCTCCAG</td><td>Biolegio</td><td></td><td>Designed by PrimerQuest tool (https://eu.idtdna.com/site)</td></tr><tr><td>Forward TBP antibody (human) reference gene&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Biolegio</td><td></td><td>Designed by PrimerQuest tool (https://eu.idtdna.com/site)</td></tr><tr><td>Reverse TBP antibody (human)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Biolegio</td><td></td><td>Designed by PrimerQuest tool (https://eu.idtdna.com/site)</td></tr><tr><td>Forward Oxa1L antibody (human) reference gene&amp;nbsp;&amp;nbsp; CACTTGCCAGAGATCCAGAAG&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Biolegio</td><td></td><td>Designed by PrimerQuest tool (https://eu.idtdna.com/site)</td></tr><tr><td>Reverse Oxa1L &amp;nbsp;antibody (human)&amp;nbsp;&amp;nbsp;&amp;nbsp; CACAGGGAGAATGAGAGGTTTATAG&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Biolegio</td><td></td><td>Designed by PrimerQuest tool (https://eu.idtdna.com/site)</td></tr><tr><td>Power SYBRGreen PCR Master Mix</td><td>Thermo Fisher Scientific</td><td>4368702</td><td></td></tr><tr><td>FACS Tubes&amp;nbsp;</td><td>Sarstedt</td><td>551578</td><td></td></tr><tr><td>MicroAmp Optical 96-Well Reaction Plate</td><td>Thermo Fisher Scientific</td><td>N8010560</td><td></td></tr><tr><td>Corning 100 mm TC-Treated Culture Dish</td><td>Corning</td><td></td><td></td></tr><tr><td>Corning Costar cell culture plates 6 well</td><td>Corning</td><td>3506</td><td></td></tr><tr><td>Refrigerated Bench-Top Microcentrifuge</td><td>Eppendorf</td><td>5415 R</td><td></td></tr><tr><td>Refrigerated Bench-Top Centrifuge Jouan CR3.12</td><td>Jouan</td><td>743205604</td><td></td></tr><tr><td>NanoDrop Lite Spectrophotometer</td><td>Thermo Scientific</td><td>ND-LITE-PR</td><td></td></tr><tr><td>BD FACSCalibur Flow Cytometer</td><td>BD Bioscience</td><td></td><td></td></tr><tr><td>QuantStudio 3 Real-Time PCR System</td><td>Thermo Fisher Scientific</td><td>A28567</td><td></td></tr></tbody></table>

studying cell cycle-regulated gene expression by two complementary cell synchronization protocols

细胞周期的基因表达程序代表了解细胞周期依赖性过程及其在疾病如癌症中的作用的关键步骤。细胞周期调节基因表达分析取决于细胞同步到特定阶段。在这里，我们描述了一种利用两种互补同步协议的方法，通常用于研究细胞周期中基因表达的周期性变化。两种方法均基于在一个限定点瞬时阻断细胞周期。羟基脲（HU）治疗的同步方案导致晚期G1 /早期S期的细胞停滞，并且从HU介导的停止释放提供通过S和G2 / M均匀进展的细胞群体。通过胸苷和诺考达唑（Thy-Noc）治疗的同步方案阻止早期有丝分裂中的细胞，并从Thy-Noc介导的停止释放提供了适合于G1期和S期的同步细胞群体尝试学习。两种方法的应用都需要监测细胞周期分布特征，这通常在细胞碘化丙啶（PI）染色后进行，流式细胞仪介导的DNA含量分析。我们表明，两个同步协议的组合使用是一种可靠的方法来清楚地确定在细胞周期中差异调节的基因（ 即 E2F1和E2F7）的转录谱，从而更好地了解它们在细胞周期中的作用流程。此外，我们显示，这种方法对于基于药物治疗（ 即丝裂霉素C，抗癌剂）的机制的研究是有用的，因为它允许区分对基因毒性因子起反应性的基因仅受细胞周期扰动影响的基因由代理人强加。

用于细胞同步的Thy-Noc和HU协议的示意图。 图1总结了U2OS细胞同步和随后的样品收集所需的步骤，以验证细胞周期的进展并进行基因表达分析。 Phospho-H3和PI染色是选择同步方法的良好评估参数。 由于培养细胞的固有异质性，必?...

Watch this Scientific Journal Video about Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols at JoVE.com

通过两种互补的细胞同步方案研究细胞周期调节的基因表达遗传学 |视频

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

我们报告两个细胞同步协议，提供一个上下文，用于研究与细胞周期的特定阶段相关的事件。我们表明，这种方法可用于分析未受扰动细胞周期中特定基因的调节，或暴露于影响细胞周期的试剂。

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 ...

studying-cell-cycle-regulated-gene-expression-two-complementary-cell

Universidad San Sebastian

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Biology

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Cellular Processes

Cell Cycle and Division

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27.1K 次观看.  University of the Basque Country, UPV/EHU. 我们报告两个细胞同步协议，提供一个上下文，用于研究与细胞周期的特定阶段相关的事件。我们表明，这种方法可用于分析未受扰动细胞周期中特定基因的调节，或暴露于影响细胞周期的试剂。 

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

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

在人原代细胞培养生物钟基因表达及荷尔蒙分泌的并行测量

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

科研

遗传学

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

单细胞基因表达利用多重RT-qPCR的表征罕见的淋巴人群的异质性

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

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 ...

生物学

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

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

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

代谢标记和流式细胞仪测定细胞周期进程的动力学

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

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

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

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

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

同步<em&gt;新月柄杆菌</em&gt;对细菌细胞周期的研究

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

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

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

This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.

细胞周期进程的时间跟踪使用流式细胞仪，无需同步

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

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

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

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

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

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

同步时间序列数据的比对以进行交叉实验比较

Alignment of Synchronized Time-Series Data Using the Characterizing Loss of Cell Cycle Synchrony Model for Cross-Experiment Comparisons

Investigating the cell cycle often depends on synchronizing cell populations to measure various parameters in a time series as the cells traverse the cell cycle. However, even under similar conditions, replicate experiments display differences in the time required to recover from synchrony and to traverse the cell cycle, thus preventing direct comparisons at each time point. The problem of comparing dynamic measurements across experiments is exacerbated in mutant populations or in alternative growth conditions that affect the synchrony recovery time and/or the cell-cycle period. 
We have previously published a parametric mathematical model named Characterizing Loss of Cell Cycle Synchrony (CLOCCS) that monitors how synchronous populations of cells release from synchrony and progress through the cell cycle. The learned parameters from the model can then be used to convert experimental time points from synchronized time-series experiments into a normalized time scale (lifeline points). Rather than representing the elapsed time in minutes from the start of the experiment, the lifeline scale represents the progression from synchrony to cell-cycle entry and then through the phases of the cell cycle. Since lifeline points correspond to the phase of the average cell within the synchronized population, this normalized time scale allows for direct comparisons between experiments, including those with varying periods and recovery times. Furthermore, the model has been used to align cell-cycle experiments between different species (e.g., Saccharomyces cerevisiae and Schizosaccharomyces pombe), thus enabling direct comparison of cell-cycle measurements, which may reveal evolutionary similarities and differences.

作者聚焦:使用表征细胞周期同步性损失模型对齐同步时间序列数据进行交叉实验比较  

One challenge of analyzing synchronized time-series experiments is that the experiments often differ in the length of recovery from synchrony and the cell-cycle period. Thus, the measurements from different experiments cannot be analyzed in aggregate or readily compared. Here, we describe a method for aligning experiments to allow for phase-specific comparisons.

使用表征细胞周期同步损失模型进行跨实验比较的同步时间序列数据比对

Investigating the cell cycle often depends on synchronizing cell populations to measure various parameters in a time series as the cells traverse the cell ...

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

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使用表征细胞周期同步损失模型进行跨实验比较的同步时间序列数据比对

使用表征细胞周期同步损失模型进行跨实验比较的同步时间序列数据比对

使用表征细胞周期同步损失模型进行跨实验比较的同步时间序列数据比对