策略跟踪Anastasis，A细胞存活的现象，细胞凋亡反转
Summary
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.
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| LSM780 confocal microscopy | Carl Zeiss | / | |
| Glass bottom culture dish | MatTek Corporation | P35G-0-14-C | |
| Transparent CultFoi | Carl Zeiss | 000000-1116-084 | |
| CO2 independent medium | Life Technologies | 18045-088 | |
| CellTracker | Life Technologies | C34552 | |
| Mitotracker Red CMXRos | Life Technologies | M-7512 | |
| Hoechst 33342 | Life Technologies | H1399 | |
| Fluorescently labeled annexin V | Biovision | K201 |
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