Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C
Department of Clinical Chemistry, Medical Spectrum Twente, Enschede, Netherlands.
J Immunol Methods. 1995 Jul 17;184(1):39-51. doi: 10.1016/0022-1759(95)00072-i.
In the early stages of apoptosis changes occur at the cell surface, which until now have remained difficult to recognize. One of these plasma membrane alterations is the translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer layer, by which PS becomes exposed at the external surface of the cell. Annexin V is a Ca2+ dependent phospholipid-binding protein with high affinity for PS. Hence this protein can be used as a sensitive probe for PS exposure upon the cell membrane. Translocation of PS to the external cell surface is not unique to apoptosis, but occurs also during cell necrosis. The difference between these two forms of cell death is that during the initial stages of apoptosis the cell membrane remains intact, while at the very moment that necrosis occurs the cell membrane looses its integrity and becomes leaky. Therefore the measurement of Annexin V binding to the cell surface as indicative for apoptosis has to be performed in conjunction with a dye exclusion test to establish integrity of the cell membrane. This paper describes the results of such an assay, as obtained in cultured HSB-2 cells, rendered apoptotic by irradiation and in human lymphocytes, following dexamethasone treatment. Untreated and treated cells were evaluated for apoptosis by light microscopy, by measuring the amount of hypo-diploid cells using of DNA flow cytometry (FCM) and by DNA electrophoresis to establish whether or not DNA fragmentation had occurred. Annexin V binding was assessed using bivariate FCM, and cell staining was evaluated with fluorescein isothiocyanate (FITC)-labelled Annexin V (green fluorescence), simultaneously with dye exclusion of propidium iodide (PI) (negative for red fluorescence). The test described, discriminates intact cells (FITC-/PI-), apoptotic cells (FITC+/PI-) and necrotic cells (FITC+/PI+). In comparison with existing traditional tests the Annexin V assay is sensitive and easy to perform. The Annexin V assay offers the possibility of detecting early phases of apoptosis before the loss of cell membrane integrity and permits measurements of the kinetics of apoptotic death in relation to the cell cycle. More extensive FCM will allow discrimination between different cell subpopulations, that may or may not be involved in the apoptotic process.
在细胞凋亡的早期阶段，细胞表面会发生一些变化，而这些变化至今仍难以识别。其中一种质膜改变是磷脂酰丝氨酸（PS）从质膜内侧转移到外层，从而使PS暴露于细胞外表面。膜联蛋白V是一种依赖Ca2+的磷脂结合蛋白，对PS具有高亲和力。因此，这种蛋白质可作为细胞膜上PS暴露的敏感探针。PS向细胞外表面的转移并非细胞凋亡所特有，在细胞坏死过程中也会发生。这两种细胞死亡形式的区别在于，在凋亡的初始阶段细胞膜保持完整，而在坏死发生的瞬间细胞膜失去完整性并变得渗漏。因此，必须结合染料排除试验来测量膜联蛋白V与细胞表面的结合，以确定细胞膜的完整性，以此作为细胞凋亡的指标。本文描述了这样一种检测方法的结果，该结果来自经辐射诱导凋亡的培养HSB - 2细胞以及地塞米松处理后的人淋巴细胞。通过光学显微镜、使用DNA流式细胞术（FCM）测量亚二倍体细胞数量以及进行DNA电泳来确定是否发生DNA片段化，对未处理和处理后的细胞进行凋亡评估。使用双变量FCM评估膜联蛋白V的结合情况，并用异硫氰酸荧光素（FITC）标记的膜联蛋白V（绿色荧光）同时评估细胞染色情况，同时进行碘化丙啶（PI）的染料排除试验（红色荧光为阴性）。所描述的检测方法可区分完整细胞（FITC - /PI - ）、凋亡细胞（FITC + /PI - ）和坏死细胞（FITC + /PI + ）。与现有的传统检测方法相比，膜联蛋白V检测方法灵敏且易于操作。膜联蛋白V检测方法能够在细胞膜完整性丧失之前检测到细胞凋亡的早期阶段，并允许测量与细胞周期相关的凋亡死亡动力学。更广泛的FCM将能够区分不同的细胞亚群，这些亚群可能参与或不参与凋亡过程。
