Fluorochrome-Labeled Inhibitors of Caspases: Convenient In Vitro and In Vivo Markers of Apoptotic Cells for Cytometric Analysis
Fluorochrome-Labeled Inhibitors of Caspases: Convenient In Vitro and In Vivo Markers of Apoptotic Cells for Cytometric Analysis
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DOI：
10.1007/978-1-60327-409-8_9
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发表时间：
2011-01-01
期刊：
影响因子：
--
通讯作者：
Johnson, Gary L.
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文献类型：
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作者：
Darzynkiewicz, Zbigniew;Pozarowski, Piotr;Johnson, Gary L.
半胱天冬酶的激活是细胞凋亡的标志。因此，开发了几种基于半胱天冬酶激活检测来识别和计数凋亡细胞频率的方法。本章描述的方法基于适用于荧光显微镜、流式细胞术和图像细胞术的荧光染料标记的半胱天冬酶抑制剂 (FLICA) 的使用。当应用于体外或体内活细胞时，用羧基荧光素或磺基罗丹明标记的细胞渗透性 FLICA 试剂专门标记正在经历凋亡的细胞。 FLICA 标记方法简单、快速、稳健，并且可以与其他细胞死亡标记物结合进行多重分析。示例包括 FLICA 与活体染色剂（碘化丙啶）结合使用、线粒体电化学势损失的检测以及使用膜联蛋白 V 荧光染料缀合物在浆细胞膜外表面暴露磷脂酰丝氨酸。细胞固定和细胞 DNA 化学计量染色后，FLICA 结合可与 DNA 倍性、细胞周期阶段、DNA 片段化和其他需要细胞透化检测的细胞凋亡事件相关联。与膜联蛋白V结合相比，FLICA检测细胞凋亡的“时间窗口”更宽，使得FLICA成为检测早期细胞凋亡的优选标记物，并且对于凋亡细胞的定量更准确。
Activation of caspases is a hallmark of apoptosis. Several methods, therefore, were developed to identify and count the frequency of apoptotic cells based on the detection of caspases activation. The method described in this chapter is based on the use of fluorochrome-labeled inhibitors of caspases (FLICA) applicable to fluorescence microscopy, and flow- and image-cytometry. Cell-permeant FLICA reagents tagged with carboxyfluorescein or sulforhodamine when applied to live cells in vitro or in vivo, exclusively label cells that are undergoing apoptosis. The FLICA labeling methodology is simple, rapid, robust, and can be combined with other markers of cell death for multiplexed analysis. Examples are presented on FLICA use in combination with a vital stain (propidium iodide), detection of the loss of mitochondrial electrochemical potential, and exposure of phosphatidylserine on the outer surface of plasma cell membrane using Annexin V fluorochrome conjugates. Following cell fixation and stoichiometric staining of cellular DNA, FLICA binding can be correlated with DNA ploidy, cell cycle phase, DNA fragmentation, and other apoptotic events whose detection requires cell permeabilization. The "time window" for the detection of apoptosis with FLICA is wider compared to that with the Annexin V binding, making FLICA a preferable marker for the detection of early phase apoptosis and more accurate for quantification of apoptotic cells.