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 border="0" cellpadding="0" cellspacing="0" width="667">
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		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Name of Material/ Equipment</td>
			<td style="width:139px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">LSM780 confocal microscopy</td>
			<td style="width:139px;">Carl Zeiss</td>
			<td style="width:127px;">/</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Glass bottom culture dish</td>
			<td style="width:139px;">MatTek Corporation</td>
			<td style="width:127px;">P35G-0-14-C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Transparent CultFoi</td>
			<td style="width:139px;">Carl Zeiss</td>
			<td style="width:127px;">000000-1116-084</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">CO2 independent medium</td>
			<td style="width:139px;">Life Technologies</td>
			<td style="width:127px;">18045-088</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">CellTracker</td>
			<td style="width:139px;">Life Technologies</td>
			<td style="width:127px;">C34552</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Mitotracker Red CMXRos</td>
			<td style="width:139px;">Life Technologies</td>
			<td style="width:127px;">M-7512</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Hoechst 33342</td>
			<td style="width:139px;">Life Technologies</td>
			<td style="width:127px;">H1399</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Fluorescently labeled annexin V</td>
			<td style="width:139px;">Biovision</td>
			<td style="width:127px;">K201</td>
			<td style="width:167px;"></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...

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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 ...

strategies-for-tracking-anastasis-cell-survival-phenomenon-that

Research

JoVE Journal

Biology

17.9K 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

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

科研

生物学

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment10. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios &lt;0.5) showing the highest occurrence10. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (&lt; 5% events with cytoplasmic PI staining).

An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.

改性膜联蛋白V /碘化丙啶检测细胞凋亡细胞死亡的准确评估

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

在早期鸡胚跟踪形态组织变形

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1.

Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

冷冻干燥细菌及其影响细胞生存的配方

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

在斑马鱼发育的细胞追踪使用Photoconvertible蛋白

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

番茄/ GFP-FLP / FRT方法马赛克成人的实时成像<em&gt;果蝇</em&gt;感光细胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

指南现代荧光定量免疫印迹与故障排除策略

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...