Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells
Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells
复制标题
DOI：
10.3791/4254
复制
发表时间：
2012-10-01
影响因子：
1.2
通讯作者：
Mariani, Francesca V.
中科院分区：
文献类型：
--
作者：
Fogel, Jennifer L.;Thu Zan Tun Thein;Mariani, Francesca V.
程序性细胞死亡（PCD）发生在成人中，以维持正常的组织稳态，并在胚胎发育期间形成组织和器官（1,2,6,7）。在发育过程中，有毒化学物质或基因改变可导致PCD增加或改变PCD模式，导致发育异常和出生缺陷（3-5）。为了了解这些缺陷的病因，胚胎研究可以与使用分化胚胎干细胞（ES）的体外试验相补充。凋亡是一种被充分研究的PCD形式，它涉及内在和外在的信号来激活caspase酶级联。特征性的细胞变化包括膜起泡、核收缩和DNA断裂。其他形式的PCD不涉及半胱天冬酶激活，可能是延长自噬的最终结果。无论PCD途径如何，死亡细胞都需要被移除。在成人中，免疫细胞执行这一功能，而在免疫系统尚未发育的胚胎中，通过另一种机制进行清除。这种机制涉及到邻近细胞（称为“非专业吞噬细胞”）承担吞噬作用——它们识别垂死细胞表面的“吃我”信号并吞噬它（8-10）。被吞噬后，碎片被带到溶酶体降解。因此，无论PCD机制如何，溶酶体活性的增加可能与细胞死亡的增加有关。为了研究PCD，在厚组织和多层分化培养物中观察溶酶体的简单实验是有用的。溶酶追踪器染料是一种高度可溶的小分子，保留在酸性亚细胞区室中，如溶酶体（11-13）。染料通过扩散和循环被吸收。由于穿透不是障碍，可见PCD在厚组织和多层培养是可能的（12,13）。相比之下，TUNEL（末端脱氧核苷酸转移酶dUTP缺口末端标记）分析（14）仅限于小样本、组织学切片和单层培养，因为该过程需要末端转移酶的进入/渗透。与苯胺蓝相比，它会扩散并被溶剂溶解，LysoTracker Red DND-99是可固定的，明亮的，稳定的。染色可以用标准荧光或共聚焦显微镜在整个安装或切片使用水或溶剂为基础的安装介质（12,13）。在这里，我们描述了使用这种染料观察正常和无超音hedgehog基因小鼠胚胎PCD的方案。此外，我们展示了PCD在分化ES细胞培养中的分析，并提出了一种简单的定量方法。综上所述，LysoTracker染色可以作为其他检测PCD方法的一个很好的补充。
Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs(1,2,6,7). During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects(3-5). 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 it(8-10). 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 lysosome(11-13). 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 possible(12,13). In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis(14), 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 media(12,13). 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.