Smolewski Piotr, Grabarek Jerzy, Halicka H Dorota, Darzynkiewicz Zbigniew
Brander Cancer Research Institute, New York Medical College, Valhalla 10595, USA.
J Immunol Methods. 2002 Jul 1;265(1-2):111-21. doi: 10.1016/s0022-1759(02)00074-1.
Activation of cysteine-aspartic acid specific proteases (caspases) in situ, in live cells, can be detected using fluorochrome-labeled inhibitors of caspases (FLICA), the reagents that covalently bind to the active center of these enzymes. In the present study, this assay was combined with a probe of plasma membrane capacity to exclude the cationic fluorochrome propidium iodide (PI). Apoptosis of HL-60 cells was induced by DNA topoisomerase I inhibitor camptothecin (CPT). The cells were then incubated with FAM-VAD-fluoro-methyl ketone (FMK), the pan-caspase FLICA, and subsequently briefly exposed to PI. The intensity of cellular green fluorescence of FLICA and red fluorescence of PI was measured by laser scanning cytometry (LSC) as well as by flow cytometry. Four distinct subpopulations were distinguished based on differences in fluorescence intensity. The subpopulations represented the sequential transitions from the stage when (a) the cells were both FLICA and PI negative (FLICA-/PI-), through the stages when (b) their caspases become progressively activated (FLICA+/PI-), (c) when their plasma membrane ability to exclude PI was lost (FLICA+/PI+), and finally (d) when the cell propensity to bind FLICA was eliminated (FLICA-/PI+). By estimating the percentage of cells in each subpopulation at different time points after administration of CPT, it was possible to study the kinetics of the transitions. The cell entry to-and progression through-these substages was asynchronous. Following this "supravital" analysis, the cells may be fixed, permeabilized, their DNA stoichiometrically stained with PI and cell cycle distribution of each of the four subpopulations analyzed. The loss of cells' ability to bind FLICA at the late stage of apoptosis indicates that caspases are either inactivated, degraded or excreted at that time point. Hence, the late apoptotic cells may not be identified solely on the evidence of the presence of activated caspases. The direct transition from FLICA-/PI- to FLICA-/PI+, bypassing the FLICA+ stages, may be considered as the marker of a primary cell necrosis.
使用荧光染料标记的半胱天冬酶抑制剂（FLICA）可原位检测活细胞中半胱氨酸天冬氨酸特异性蛋白酶（caspases）的激活情况，这些试剂可与这些酶的活性中心共价结合。在本研究中，该检测方法与质膜容量探针相结合，以排除阳离子荧光染料碘化丙啶（PI）。DNA拓扑异构酶I抑制剂喜树碱（CPT）诱导HL-60细胞凋亡。然后将细胞与泛半胱天冬酶FLICA FAM-VAD-氟甲基酮（FMK）孵育，随后短暂暴露于PI。通过激光扫描细胞术（LSC）和流式细胞术测量FLICA的细胞绿色荧光强度和PI的红色荧光强度。根据荧光强度的差异区分出四个不同的亚群。这些亚群代表了从（a）细胞同时为FLICA和PI阴性（FLICA-/PI-）阶段开始的连续转变，经过（b）其半胱天冬酶逐渐激活（FLICA+/PI-）阶段、（c）其质膜排除PI的能力丧失（FLICA+/PI+）阶段，最后到（d）细胞结合FLICA的倾向消除（FLICA-/PI+）阶段。通过估计CPT给药后不同时间点每个亚群中细胞的百分比，可以研究转变的动力学。细胞进入和通过这些亚阶段是异步的。在进行这种“超活”分析后，可以固定细胞、使其通透，用PI对其DNA进行化学计量染色，并分析四个亚群中每个亚群的细胞周期分布。凋亡后期细胞结合FLICA能力的丧失表明此时半胱天冬酶要么失活、降解，要么被排出。因此，晚期凋亡细胞可能不能仅根据激活的半胱天冬酶的存在来鉴定。从FLICA-/PI-直接转变为FLICA-/PI+，绕过FLICA+阶段，可被视为原发性细胞坏死的标志。
