Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kiev, 01030, Ukraine,
Cytotechnology. 2013 Mar;65(2):157-72. doi: 10.1007/s10616-012-9481-y. Epub 2012 Jul 14.
Dramatic changes in the structure of cell membranes on apoptosis allow easy, sensitive and non-destructive analysis of this process with the application of fluorescence methods. The strong plasma membrane asymmetry is present in living cells, and its loss on apoptosis is commonly detected with the probes interacting strongly and specifically with phosphatidylserine (PS). This phospholipid becomes exposed to the cell surface, and the application of annexin V labeled with fluorescent dye is presently the most popular tool for its detection. Several methods have been suggested recently that offer important advantages over annexin V assay with the ability to study apoptosis by spectroscopy of cell suspensions, flow cytometry and confocal or two-photon microscopy. The PS exposure marks the integrated changes in the outer leaflet of cell membrane that involve electrostatic potential and hydration, and the attempts are being made to provide direct probing of these changes. This review describes the basic mechanisms underlying the loss of membrane asymmetry during apoptosis and discusses, in comparison with the annexin V-binding assay, the novel fluorescence techniques of detecting apoptosis on cellular membrane level. In more detail we describe the detection method based on smart fluorescent dye F2N12S incorporated into outer leaflet of cell membrane and reporting on apoptotic cell transformation by easily detectable change of the spectral distribution of fluorescent emission. It can be adapted to any assay format.
细胞膜在细胞凋亡过程中的结构发生剧烈变化，这使得可以使用荧光方法轻松、灵敏且无损地分析这个过程。活细胞中存在强烈的质膜不对称性，其在凋亡过程中的丧失通常可以通过与磷脂酰丝氨酸（PS）强烈且特异性相互作用的探针来检测。这种磷脂变得暴露在细胞膜表面，目前应用荧光染料标记的 annexin V 是检测 PS 暴露的最常用工具。最近提出了几种方法，这些方法通过细胞悬浮液的光谱学、流式细胞术和共聚焦或双光子显微镜对细胞凋亡进行研究，提供了比 annexin V 测定法更重要的优势。PS 暴露标志着细胞膜外层的整体变化，涉及静电势和水合作用，目前正在尝试提供对这些变化的直接探测。这篇综述描述了细胞凋亡过程中细胞膜不对称性丧失的基本机制，并与 annexin V 结合测定法进行了比较，讨论了在细胞膜水平上检测细胞凋亡的新型荧光技术。我们更详细地描述了一种基于智能荧光染料 F2N12S 的检测方法，该染料整合到细胞膜的外层，通过荧光发射光谱分布的容易检测到的变化来报告凋亡细胞的转化。它可以适应任何测定格式。
