Name: strategies for tracking anastasis, a cell survival phenomenon that reverses apoptosis
Uploaded: 2015-02-16T13:00:00.000+00:00
Duration: 12 min 55 s
Description: Watch this Scientific Journal Video about Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis at JoVE.com

我们感谢牧师博士拉尔夫Bohlmann和牧师詹姆斯Voelz博士提出了这个词"anastasis"来形容逆转细胞凋亡;道格拉斯·R·格林对HeLa细胞稳定表达细胞色素C-GFP;查尔斯·M.·鲁丁和Eric E.加德纳的H446细胞;希瑟羔羊，在视频卡通画援助;怡辉杨本手稿的价值的讨论。这项工作是由尤德爵士奖学金（HLT），博士沃尔特·司徒纪念奖学金（HLT），富布赖特资助007-2009（HLT），生命科学研究基金会的奖学金（HLT）的支持下，美国国立卫生研究院授予NS037402（JMH）和NS083373（江铃控股），以及香港大学教育资助委员会的AoE / B-07/99（MCF）。何林堂是生命科学研究基金会的Shurl和凯库尔奇基金会研究员。

1.准备细胞活细胞成像的<ol><li>为了方便的形态变化的检测，选择贴壁细胞例如HeLa（人宫颈​​癌）细胞是平坦的基板上，以更好地可视化改变质膜和细胞器的。 注:细胞凋亡的逆转已在各种哺乳动物细胞中12,13，包括原代小鼠肝细胞，原代巨噬细胞，原代大鼠心肌细胞中观察到，并且还细胞系，例如人胚胎肾HEK-293T细胞，非洲绿猴肾上皮的COS -7细胞，小鼠心肌的HL-1细胞，小鼠NIH 3T3成纤维细胞，雪貂（ 鼬putoris呋喃 ）脑CRL1656细胞，人类皮肤癌A375细胞，人睾丸癌CRL1973细胞，人小细胞肺癌H446细胞，人肝癌HepG2细胞，人乳腺癌MCF7细胞，人前列腺癌PC3细胞，和人神经母细胞瘤SH-SY5Y细胞。 </li><li>的玻璃底细胞培养皿以用于清洁和灭菌的无水乙醇预洗涤盖玻片。 注1:使用的玻璃底培养皿被推荐用于高质量微分干涉对比（DIC）显微镜，和高放大倍数的共焦或落射荧光显微镜（见讨论）。 注2:对于高放大倍率（40X，60 / 63X或100X的目标，高数值孔径1.3或1.4），使用的细胞培养皿更薄的玻璃底部（厚度0.085-0.16毫米），提供更长的工作距离成像（见协议3 ）。 </li><li>取决于细胞类型，玻璃底培养皿可能需要涂覆有聚-D-赖氨酸（0.1毫克/毫升），胶原蛋白（在0.01M的乙酸0.01％溶液）和/或纤连蛋白（5微克/毫升）之前播种细胞。 注意:需要对不同的细胞系的类型和涂布剂的浓度的优化。 HeLa细胞可以直接在玻璃上附着无涂层。然而，一些细胞，苏CH小鼠心肌的HL-1细胞，不能直接附着于玻璃表面，但需要特定的培养和涂层协议37。 </li><li>种子〜2-3×10 5个细胞在35 mm预涂覆的玻璃底部的细胞培养皿中，并在37摄氏度（℃），用5％的CO 2，每天达到80％汇合。 注1:细胞密度，孵育时间，和培养条件的最佳条件可以变化，这取决于细胞类型。细胞汇合可以影响细胞（见方案2）的凋亡反应。在高汇合，细胞可以是细胞凋亡刺激的相同浓度的较不敏感。 注2:避免过度融合细胞，为高密度可以掩盖单个细胞和细胞器的形态变化。 </li></ol> 2.应用和删除凋亡细胞的刺激<ol><li>不久在使用前，用37℃的细胞培养液中预混合的细胞凋亡诱导剂。乙醇（3.6-4.5％体积/体积）作为细胞凋亡诱导剂，在本示范。 注1:我们的公布和未公布的数据表明，细胞也可逆转是受其他凋亡刺激12,13触发凋亡，如二甲亚砜（DMSO，8％体积/体积为24小时），星形孢菌素（STS，0.5微米为1小时），和紫杉醇（1μM12小时）。需要优化剂量的用于触发凋亡为不同类型的细胞，以达到可触发大部分细胞在人口中进行细胞凋亡的最低剂量。 注2:预混合的细胞凋亡诱导剂具有细胞培养基是重要，以避免细胞的不均匀接触凋亡刺激。 </li><li>图像的一组健康的细胞在细胞培养皿的施加的凋亡诱导剂来建立基线形态之前。 </li><li>除去从细胞培养皿原始介质，然后应用2毫升预混细胞凋亡诱导剂的菜触发细胞凋亡。孵育所述细胞在37℃，5％的CO 2（或其它正常培养条件）。 </li><li>孵育后，观察细胞的光，落射荧光或共聚焦显微镜来监测凋亡特征的进展。 注1:细胞经历凋亡，将显示可以由显微镜和基于荧光的生物传感器来检测凋亡的形态学，生物化学和分子标志（见议定书3和4）。 注2:细胞不同的细胞凋亡诱导剂的孵育时间会有所不同，这取决于细胞类型，细胞的条件（如汇合和营养状态），和死亡的刺激的施加剂量。小心滴定可能需要。 </li><li>当细胞显示凋亡的标志，通过用温热（37℃，或其他正常培养温度）新鲜细胞培养基一次洗涤细胞除去细胞凋亡诱导剂。 注1:由于凋亡细胞是较为松散的连接培养板，以应用和轻轻取出介质，使得细胞不会被冲走是重要的。 注2:在线悬浮的细胞可以从上清液通过温和离心（160×g离心1分钟）来回收，并重新添加到培养皿。 </li><li>孵育细胞，在37℃用2ml /新鲜细胞培养基在5％CO 2 35毫米的菜。可替代地，使用的条件培养基（从健康细胞中收集无细胞的培养基中），以进一步增强凋亡细胞的存活。 注意:洗涤和温育的细胞用新鲜培养基使大多数细胞乙醇暴露12,13后逆转乙醇诱导的凋亡。然而，重复的，可能需要此洗涤步骤时，细胞暴露于其它细胞凋亡刺激（见讨论）。 </li></ol> 3.活细胞显微镜<ol><li>我们建议使用一个倒置的共焦或落射荧光显微镜环境控制（37°C，5％的CO 2），用于活细胞显微镜检查。 注意:也可以将图像的使用直立显微镜用与水浸渍目标细胞。直接蘸目标到培养基中图像的细胞。然而，细胞可以通过浸渍物镜聚焦过程中压碎。 </li><li>预暖显微镜（如环境控制室，舞台孵化器，客观加热器）成像前至少2小时。 注意:这允许显微镜部件达到热平衡，这样它可避免焦点和xy平面的偏移的漂移，由于各组分的热膨胀和收缩。 </li><li>放置培养细胞的35毫米的玻璃底培养皿（参见方案1）在倒置显微镜的阶段，并用40X或63X计划复消色差透镜物镜具有的数值孔径（NA）为1.3或1.4捕获细胞图像，用于从成像单元透过玻璃盖玻片的菜底部。 注意:避免将超过200毫升培养基到35毫米的玻璃底皿中，作为介质的重量会导致玻璃底的曲率，使得难以图像单元的字段在同一焦平面上。 </li><li>在整个成像过程中保持细胞在37℃或相应的正常温度。 注:细胞凋亡的方法，和细胞凋亡的可能逆转，取决于温度敏感酶活性。因此，在37℃下保持的细胞（ 例如，用显微镜阶段顶部孵化）是在整个实验重要。降低温度能减缓细胞凋亡反应和清除细胞凋亡刺激后恢复响应。 </li><li>使用湿度设备或躺在一个透明的箔（见材料）上的培养皿中蒸发介质，以减少水分流失。 注:箔可能会破坏光的极性DIC显微镜。通过调整偏振片在光路恢复极性。 </li><li>保持pH值在细胞培养基（pH为6.8 -7.3）孵育在5％CO 2在显微镜的环境控制腔室。 注意:维护在培养基中的pH值可以通过添加HEPES缓冲剂来达到的，或者通过使用商用CO 2独立培养基（见材料）。最佳条件可以变化，这取决于细胞类型。 </li><li>在成像过程中最小化荧光/激光（激发光强度）暴露于细胞，以避免光毒性通过降低的荧光强度为最低，以获得细胞/亚细胞结构或表达的生物传感器（在协议4详细内容）的高品质的图像必需的。 </li></ol>期间和之后细胞凋亡活动4.策略检测和跟踪Anastasis <ol><li>质膜出泡，细胞质凝聚，细胞皱缩和凋亡小体的形成（参见图1A - C，E）。 <ol><li>执行时间推移活细胞迪菲rential干涉对比（DIC），或相差显微镜跟踪一组健康细胞，并观察其细胞形态（见协议3的活细胞显微镜，并见讨论）。 注1:减少的光源，用于DIC /相位合同成像强度，以避免光毒性的细胞。 注2:如果DIC和相差显微镜不可用，则使用CellTracker染色胞浆勾勒活细胞的形态为共焦或落射荧光显微镜和监测细胞形态​​。 </li><li>应用细胞死亡的刺激触发细胞凋亡（参见协议2的应用程序，并清除凋亡刺激）。 </li><li>观察处理的细胞的细胞凋亡的形态学特点如质膜起泡，细胞质凝聚，细胞皱缩和细胞凋亡体的形成（ 图1A，1B，1C，1E）。 </li><li>洗去死亡的刺激，并重新供应细胞与细胞时新鲜培养基DISPLA细胞凋亡形态Ÿ标志。 注1:应用细胞死亡的刺激或在时间推移活细胞成像改变显微镜舞台上的细胞培养液中可以通过使用灌注细胞培养室来实现，也可以直接在细胞培养皿中不小心触碰进行吹打盘中，中成像之间的时间间隔。 注2:使用焦点漂移补偿系统，以避免出由于显微镜系统的热平衡的损失的细胞的焦点的改变的细胞培养基（见讨论）之后。 </li><li>持续时间推移成像追踪细胞凋亡显示的标志的命运。要彻底扭转凋亡细胞可以修复损伤，恢复正常的形态持平（ 图1A，1B，1C，1E）。 </li></ol></li><li>线粒体分裂，DNA /染色质浓缩和核碎裂（ 见图1D，1F，1G）。 <ol><li>可视化和线粒体RIA，染色细胞用50nM的MitoTracker红/深红/绿荧光染料，并同时染色用的Hoechst 33342蓝色核染料在培养基中10μg/ ml的20分钟的核，在37℃，5％的CO 2。 注1:减少染料和孵育时间的浓度，以避免细胞毒性和降低背景荧光时的需要。优化染色条件，得到了良好的信噪比与染料和温育时间为染色的最小量。 注2:使用荧光染料的线粒体，如MitoTracker红CMXRos，MitoTracker深红FM，或MitoTracker绿色FM，从线粒体细胞凋亡过程中不会泄露出去。这使得细胞凋亡和anastasis在可视化线粒体形态。请参阅材料和设备表中有关这些污渍的详细信息。 </li><li>保持染色的细胞在黑暗中，以避免光漂白。 </li><li>由洗涤细胞去除多余的污点3次，用37℃的磷酸盐缓冲盐水（PBS）溶液或新鲜的细胞培养液中，用1分钟温育在每次洗涤之间的新鲜培养基，然后在最后一次洗涤之前进一步孵育在新鲜培养基中20分钟，以允许过大的渍退出细胞减少非特异性背景信号进行成像。 注意:缩短培养时间与细胞培养基后染色可能导致高背景用于成像。 </li><li>前凋亡​​诱导施加执行实时活细胞共聚焦或落射荧光显微镜图像的健康细胞（参见方案3为活细胞显微术）。 注:健康的细胞显示管状线粒体和细胞核周围的大多数细胞类型（ 图1D，1F，1G）。 </li><li>触发细胞凋亡（参见方案2为应用和去除凋亡刺激向细胞）。 </li><li>持续时间推移活细胞显微镜观察线粒体和核形态学改变IES后立即死亡刺激被施加到细胞。观察细胞显示凋亡的标志，如线粒体碎片和肿胀，染色质凝聚和核碎裂，而相比之下，健康细胞显示管状线粒体和圆形核。 </li><li>当细胞显示凋亡的标志，洗净，用新鲜培养基供给细胞，并继续延时成像追踪细胞的命运。要彻底扭转凋亡细胞恢复正常的线粒体和核形态（ 图1D，1F，1G）。 注意:在并行执行的DIC显微镜时可能获得的附加信息通过观察细胞形态的改变，在同一组中的细胞（参见协议4.1 DIC显微镜）的访问凋亡的阶段。 </li></ol></li><li>检测细胞色素线粒体释放c。使用细胞稳定表达细胞色素C-GFP融合公关otein（参见图2）。 <ol><li>染色稳定表达细胞色素C-GFP 23,24与MitoTracker红色标记极化细胞的线粒体（参见4.2.1步骤4.2.3活细胞线粒体染色）。 </li><li>执行实时活细胞共焦或落射荧光显微镜追踪健康细胞（在协议3的活细胞成像细节）。 注:细胞色素C-GFP信号主要定位于健康细胞（ 图2A I，2B）的线粒体。 </li><li>触发细胞凋亡（参见方案2为应用和去除凋亡刺激向细胞）。持续时间推移显微镜观察细胞色素C-GFP易位从线粒体到细胞质（ 图2Aii-III，2B）。 注意:线粒体释放细胞色素c到胞质溶胶是线粒体外膜透化的标记物（MOMP）4，在线粒体依赖性凋亡21,22的关键步骤</li><li>当细胞显示在细胞质中的细胞色素C-GFP信号，去除细胞死亡的刺激和供应电池用新鲜培养基（见协议2）。持续时间推移成像追踪细胞胞浆GFP的命运。 注:反向凋亡细胞恢复正常的细胞形态，并可能是通过降解或易位减少细胞内细胞色素C-GFP信号反馈到线粒体（ 图2Aiv-VII，2B）。 </li><li>捕获的DIC图像在平行于获得附加信息用于通过在单细胞或细胞群（参见协议4.1 DIC显微镜）细胞形态观察改变访问凋亡的阶段。 </li></ol></li><li>检测使用胱天蛋白酶的生物传感器caspase活性的（ 见图3） <ol><li>转染的细胞与胱天蛋白酶的生物传感器NES-DEVD-YFP-NLS为16至24小时对他们进行细胞凋亡刺激13之前。 </li><li>染色染培养用Hoechst 33342标记细胞的细胞核（参见4.2.1步骤4.2.3活细胞核染色）。 </li><li>执行实时活细胞共焦或落射荧光显微镜追踪健康细胞（在协议3的活细胞成像细节）。 注意:NES-DEVD-YFP-NLS生物传感器信号，主要定位于由于其核输出信号（NES）（ 图3A，3Bi和3C，见讨论）的健康细胞的胞质溶胶中。 </li><li>触发细胞凋亡（参见方案2为应用和去除凋亡刺激向细胞）。持续时间推移显微镜观察YFP核易位。 注:半胱氨酸蛋白酶切割激活的DEVD主题的蛋白酶生物传感器NES-DEVD-YFP-NLS，导致YFP-NLS易位从细胞质到细胞核（ 图3A，3Bii-IV，见讨论）。 </li><lI&gt;当细胞显示核YFP，除去细胞凋亡刺激和供应电池用新鲜培养基（见协议2）。 注意:反向凋亡恢复正常的细胞形态的细胞，但保留核YFP的信号（ 图3BV-X，小区1和2，和3D）。 </li><li>持续时间推移成像跟踪细胞显示核YFP的命运。 注:核YFP信号将成为降级的几个小时后，细胞已经逆转细胞凋亡（ 图3B，VI-X，单元1，见结果与讨论）大概是通过细胞的正常通关机制，如蛋白酶体降解。 </li><li>捕获的DIC图像在平行于获得附加信息用于通过在细胞形态在单细胞或一组细胞观察改变访问凋亡的阶段。 （见协议4.1 DIC显微镜）。 </li></ol></li><li>磷脂酰丝氨酸的细胞表面暴露（参见图4） <ol><li>在GL种子细胞屁股盖玻片达到80％细胞汇合（参见协议1）。 </li><li>联合染色细胞红色荧光MitoTracker和蓝色的Hoechst 33342染色的线粒体和细胞核，分别为（参见4.2.1步骤4.2.3活细胞线粒体和细胞核染色）。 </li><li>触发细胞通过使用细胞凋亡诱导发生凋亡（参见方案2）。 </li><li>应用荧光标记的膜联蛋白V（参见材料）在37℃下除去细胞凋亡诱导剂，以检测凋亡细胞的表面上的磷脂酰丝氨酸的暴露前进行染色的凋亡细胞10分钟。 注1:健康细胞不染色膜联蛋白V，因为磷脂通常螯合上的内小叶的细胞质膜38。凋亡细胞被染色，膜联蛋白V，其结合到磷脂酰丝氨酸在细胞表面上（ 图4A和4B）。 注2:荧光标记的膜联蛋白V，也可以适用于染色的凋亡细胞IMM除去细胞凋亡诱导剂，用10分钟温育后ediately。 </li><li>除去细胞死亡刺激物，用温PBS或新鲜培养基一次洗涤染色的细胞，然后在37℃下供给的细胞用新鲜的培养基进行2小时，用5％的CO 2（见方案2）。 注1:细胞逆转细胞凋亡恢复正常的形态和恢复正常的形态后保留荧光标记的膜联蛋白V信号（ 图4A和4B）。 注2:随着时间的推移对回收的细胞（见讨论）的膜联蛋白V信号减小。 </li><li>固定细胞，用4％多聚甲醛含8％蔗糖在1×PBS中进行20分钟，在黑暗中在室温下选择的时间点，并清洗所述固定的细胞用PBS进行3次在载玻片上固定的细胞之前去除低聚甲醛盖玻片与抗褪色封固剂（见材料）的共焦或落射荧光显微镜和清除细胞凋亡诱导剂后，观察细胞的膜联蛋白V。 注1:蔗糖固定液固定期间保持细胞膜结构。 注2:存储在黑暗的多聚甲醛溶液在4℃，以防止退化。 注3:将它应用到细胞固定，以避免冷休克到细胞中之前预热多聚甲醛溶液至室温。 </li></ol></li></ol>

The authors have nothing to disclose.

Anastasis指现象，已激活的细胞死亡途径细胞随后扭转死亡过程和生存。在这里，我们已经证明，活细胞成像，可用于确认同一个体细胞其实可以在后期阶段逆转细胞凋亡的过程，然后继续生存和繁殖。我们的协议描述了几种优化的小区类型特定的处理条件，以诱导细胞凋亡和使细胞的相当大的比例，以进行细胞凋亡的逆转与几个生物标记进行验证监控。关于技术问题，玻璃底培养皿使用，因为高...

细胞凋亡（希腊的"坠落死亡"）被普遍认为是一个单向的过程，细胞自杀1-7结束。亲死亡的基因产生额外的电池，否则死在整体动物，包括细胞已经开始凋亡通路8,9的生存基因的破坏。同样，基因操作使健康的哺乳动物细胞人为显示"吃我"的信号，或者说失去粘附性的细胞外基质难逃一死的全细胞吞噬或entosis期间，分别为10,11。然而，我们和其他人已经表明，如果没有遗传操作正常健康的哺乳动物细胞和细胞系还可以从细胞凋亡12-15的早期阶段中恢复。使用工具来追踪单个细胞，我们进一步证明凋亡12,13的后期恢复，细胞已通过检查站的重要典型的后LY标记"不归路"2-6。晚期凋亡的这些检查点包括线粒体释放细胞色素c，活化胱天蛋白酶的，核碎裂和凋亡小体形成。我们通过一个希腊复合词"anastasis"，意思是"上升 ​​到生活"，来形容这种逆转细胞凋亡的细胞死亡2-6的边缘。 除非整个垂死恢复过程是由活细胞成像观测，它是具有挑战性的区分也从没有经历过的事件凋亡细胞发生anastasis细胞。几十年来的工作人士透露，细胞自杀凋亡的形态特征是由进化上保守的生化和分子事件16-19驱动。这些事件促使自我毁灭的细胞通过消除受损或D，有利于在单细胞和多细胞生物的发育和homoeostatic流程angerous细胞16-19。而凋亡细胞可以通过细胞凋亡1,5,6,16,20标准化形态学，生物化学和分子表现容易地区别，目前有特定于anastasis 12,13没有已知的标记物。重要的是，经历了anastasis细胞似乎是正常的健康细胞，而细胞刚刚开始扭转凋亡出现凋亡死亡的细胞12,13。因此，新的工具来完成与给定的细胞存活此前曾经历了积极的凋亡过程肯定需要的。 凋亡通常假设为一个不可逆的级联，因为它是一种快速，大规模破坏的过程。而它可能需要几分钟到几天对某些细胞以引发细胞凋亡，一旦线粒体已经发布凋亡因子如细胞色素C进入细胞质21,22，胱天蛋白酶可以在5分钟23,24内被激活，接着细胞质和在10分钟内25-27核缩合，细胞死亡，此后不久25-27。激活胱天蛋白酶裂解和失活对蜂窝拆卸2,28，目的关键的结构和功能部件，如内切酶抑制剂DFF45 / ICAD 29,30编排凋亡。半胱氨酸蛋白酶还能激活促凋亡因子，如BCL-2家族成员BID，其中转运至线粒体，促进细胞色素线粒体释放C. 31,32。蛋白酶活性也导致磷脂酰丝氨酸的细胞表面暴露作为一个"吃我"的信号，促进通过吞噬垂死33细胞巨噬细胞或相邻细胞吞噬。此外，细胞凋亡事件使线粒体功能失调，破坏细胞的生物能学和新陈代谢34,35,36。因此，从这种破坏复苏看起来可能性不大直观。 相反，原来的期望ations，细胞甚至可以在后期阶段扭转凋亡细胞死亡的过程。通过连续地监测在培养濒死细胞的命运，我们观察到晚期凋亡的可逆性的范围内的初级细胞和细胞系12,13。除去死亡的刺激的允许回收从细胞凋亡的显性功能，如线粒体碎裂，染色质凝聚，DNA损伤，质膜起泡，磷脂酰丝氨酸的细胞表面暴露，线粒体细胞色素c释放，caspase激活，核碎裂，细胞皱缩，和凋亡小体形成。这些观察结果提出有关的功能，后果和anastasis机制悬而未决的问题。为了解决这些问题，一个先决条件是要可靠地识别已发生anastasis细胞。在这里，我们描述了活显微镜的方法和胱天蛋白酶的生物传感器，用于检测细胞先前已逆转晚期凋亡和第恩幸存。

<table><tbody><tr><td>LSM780 confocal microscopy</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Glass bottom culture dish</td><td>MatTek Corporation</td><td>P35G-0-14-C</td><td></td></tr><tr><td>Transparent CultFoi</td><td>Carl Zeiss</td><td>000000-1116-084</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; independent medium</td><td>Life Technologies</td><td>18045-088</td><td></td></tr><tr><td>CellTracker</td><td>Life Technologies</td><td>C34552</td><td></td></tr><tr><td>Mitotracker Red CMXRos</td><td>Life Technologies</td><td>M-7512</td><td></td></tr><tr><td>Hoechst 33342</td><td>Life Technologies</td><td>H1399</td><td></td></tr><tr><td>Fluorescently labeled annexin V</td><td>Biovision</td><td>K201</td><td></td></tr></tbody></table>

strategies for tracking anastasis, a cell survival phenomenon that reverses apoptosis

Anastasis（希腊的"上升到生命"）是指垂死细胞的恢复。之前，这些细胞恢复，它们通过细胞凋亡的重要的检查站，包括线粒体碎裂，释放线粒体细胞色素C进入细胞质，活化胱天蛋白酶的，染色质凝集，DNA损伤，核碎裂，细胞膜起泡，细胞收缩，细胞表面暴露已经过去了磷脂酰丝氨酸的，和凋亡小体形成。当凋亡刺激被死前删除，从而使死亡的细胞逆转细胞凋亡和潜在的其他死亡机制Anastasis可能发生。因此，anastasis似乎涉及生理愈合过程也可以维持受损细胞不适当。的功能和anastasis的机制仍不清楚，阻碍在部分由有限的工具为明显健康细胞的恢复之后检测过去的事件。策略来检测anasta矽统将使生理机制的研究，不死细胞疾病的病理的危害和潜在的治疗方法来调节anastasis。在这里，我们将介绍使用活细胞显微镜和哺乳动物蛋白酶生物传感器的识别和跟踪anastasis在哺乳动物细胞中有效的策略。

研究细胞凋亡的逆转，组织培养细胞的第一次接触到死亡刺激触发细胞凋亡。当细胞凋亡显示的标志，新鲜的培养液中，然后应用到洗去刺激，然后孵化死亡的细胞，使恢复（ 图1A）。这里，正在解决的关键问题是个人的濒死细胞培养多远，能对细胞凋亡的进展，仍然接受anastasis。这个问题可以明确回答连续监测的生物标记物来跟踪使用下面描述的方法的细胞命运。 <p class="jove_con...

Watch this Scientific Journal Video about Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis at JoVE.com

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

策略跟踪Anastasis，A细胞存活的现象，细胞凋亡反转

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

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18.0K 次观看.  Johns Hopkins University Bloomberg School of Public Health. The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis. 

视频: Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

长期活细胞成像对紫杉醇反应细胞命运的评估

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

科研

癌症研究

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

细胞凋亡逆转后新形成乳腺癌干细胞样细胞的流式细胞仪检测

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer treatments as used in clinical oncology. It enables a quantitative and longitudinal analysis of the DNA damage response after genotoxic stress, as induced by radiotherapy and a multitude of anti-cancer drugs. The method covers all stages of the DNA damage response, providing endpoints for induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and cell death by apoptosis in case of repair failure. Combining these measurements provides information about cell cycle-dependent treatment effects and thus allows an in-depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. As the effect of many cancer therapeutics including chemotherapeutic agents and ionizing radiation is limited to or strongly varies according to specific cell cycle phases, correlative analyses rely on a robust and feasible method to assess the treatment effects on the DNA in a cell cycle-specific manner. This is not possible with single-endpoint assays and an important advantage of the presented method. The method is not restricted to any particular cell line and has been thoroughly tested in a multitude of tumor and normal tissue cell lines. It can be widely applied as a comprehensive genotoxicity assay in many fields of oncology besides radio-oncology, including environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells.

The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.

细胞周期特异性测量βH2AX和细胞凋亡后热源毒性应力通过流细胞测定

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

透过玻璃看:时间推移显微镜和单细胞的纵向跟踪研究抗癌治疗

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

医学

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

用 CaspaseTracker 生物传感器检测 Anastasis体内

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

使用LysoTracker检测及鉴别胚胎干细胞在胚胎细胞程序性死亡

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

生物学

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9.
The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.

E4orf4诱导的细胞死亡的击倒效果的基因表达和测量条件击倒细胞系的制备

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various ...

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 ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

的细胞内信号与相邻小区发生细胞凋亡通过互动活细胞诱导活动标识

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

An Introduction to Cell Death

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as cell injury, low nutrient levels, or signaling proteins. Whereas necrosis is considered to be an &#8220;accidental&#8221; or unexpected form of cell death, evidence exists that apoptosis and autophagy are both programmed and &#8220;planned&#8221; by cells.In this introductory video, JoVE highlights key discoveries pertaining to cell death, including recent work done in worms that helped identify genes involved in apoptosis. We then explore questions asked by scientists studying cell death, some of which look at different death pathways and their interactions. Finally, several methods to assess cell death are discussed, and we note how researchers are applying these techniques in their experiments today.

Cell Death: Apoptosis, Autophagy and Necrosis

导论细胞死亡

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as ...