Ni J, Watson J V, Cox H, Karpas A
Department of Haematology, University of Cambridge, England.
Cytometry. 1993;14(3):281-6. doi: 10.1002/cyto.990140308.
An improved twin-probe multiparameter flow cytometric technique was applied to examine a novel cytotoxin, Factor (F2), induced tumor cell permeability. Ability to retain preloaded intracellular bis-carboxyethyl carboxyfluorescein (BCECF, green fluorescence) and to exclude extracellular propidium (red fluorescence) was measured simultaneously with forward and right-angle scatter. In addition to the two expected cell populations which were stained green negative, red positive ("membrane-damaged" and "non-viable", Region 2), and green positive, red negative ("membrane intact" and "viable", Region 3), a third population was seen which fluoresced neither green nor red and displayed intermediate light scatter characteristics (Region 1). K562 cells progressed from Region 3 to Region 1, and then from Region 1 to Region 2 after treatment with F2. These results suggest that sequential changes in membrane structure lead to increased permeability, first with respect to intracellular BCECF and then in turn to extracellular propidium. Flow cytometric changes caused by F2 were detectable 10 min after treatment with 2.5 U/ml of F2, and 5 min after 10 or 40 U/ml of F2. Flow cytometric analysis showed that F2-induced tumor cell lysis and growth inhibition were accompanied by rapid alternations in tumor cell membrane permeability. Flow cytometric analysis also distinguished F2 cytotoxicity from phorbol myristate acetate (PMA) associated cytotoxicity to K562 cells and determined that F2 produced spontaneously or induced by PMA and/or ciprofloxacin had a similar ability to induce tumor cell membrane permeability change.
一种改进的双探针多参数流式细胞术被应用于检测一种新型细胞毒素——因子（F2）诱导的肿瘤细胞通透性。通过前向散射和直角散射同时测量细胞保留预加载的细胞内双羧乙基羧基荧光素（BCECF，绿色荧光）和排除细胞外碘化丙啶（红色荧光）的能力。除了两个预期的细胞群体，即绿色阴性、红色阳性（“膜损伤”和“非存活”，区域2），以及绿色阳性、红色阴性（“膜完整”和“存活”，区域3）之外，还观察到第三个群体，其既不发出绿色荧光也不发出红色荧光，并表现出中等的光散射特征（区域1）。用F2处理后，K562细胞从区域3进展到区域1，然后从区域1进展到区域2。这些结果表明，膜结构的顺序变化导致通透性增加，首先是对细胞内BCECF而言，然后依次是对细胞外碘化丙啶而言。用2.5 U/ml的F2处理10分钟后，以及用10或40 U/ml的F2处理5分钟后，就可以检测到F2引起的流式细胞术变化。流式细胞术分析表明，F2诱导的肿瘤细胞裂解和生长抑制伴随着肿瘤细胞膜通透性的快速改变。流式细胞术分析还区分了F2对K562细胞的细胞毒性与佛波酯（PMA）相关的细胞毒性，并确定F2自发产生的或由PMA和/或环丙沙星诱导产生的具有类似的诱导肿瘤细胞膜通透性变化的能力。
