Michie J, Akudugu J, Binder A, Van Rensburg C E, Böhm L
Department of Radiation Oncology, Radiobiology Laboratory, Department of Pharmacology, University of Stellenbosch, Faculty of Health, Tygerberg Hospital, P.O. Box 19063, Tygerberg 7505, South Africa.
Anticancer Res. 2003 May-Jun;23(3B):2675-9.
Determination of the drug concentration required to kill 50% of the tumour cells (EC50) does not take into account the propensity of cells to undergo apoptosis and necrosis. These 2 parameters and the viable cells are here assessed by a flow cytometric (FC) approach using propidium iodide (PI) and FITC-Annexin V staining.
A number of carcinoma cell lines of defined p53 status were exposed to cis-PtII for 24 hours, stained with PI and FITC-Annexin V and analyzed by FC. Unstained viable cells, early apoptotic cells and necrotic cells were scored separately in dual parameter plots of green fluorescence (FITC) against red fluorescence (PI) to generate dose response curves.
EC50 values for cell viability were found to be 1-4 times higher than survival data from colony assays resembling data obtained by MTT or Crystal Violet vital dye staining. Percentage apoptosis measured by Annexin V binding was in agreement with microscopic scoring of apoptotic cells after Acridine Orange staining.
The FC assay described gives a good estimate of cell viability resembling data from vital dye staining assays and provides additional information on apoptosis and necrosis. FC data from Annexin V binding and microscopic scoring after Acridine Orange staining were in excellent agreement.
确定杀死50%肿瘤细胞所需的药物浓度（EC50）未考虑细胞发生凋亡和坏死的倾向。这里通过使用碘化丙啶（PI）和异硫氰酸荧光素标记的膜联蛋白V（FITC-Annexin V）染色的流式细胞术（FC）方法评估这两个参数以及活细胞。
将多种已知p53状态的癌细胞系暴露于顺铂II 24小时，用PI和FITC-Annexin V染色并通过FC分析。在绿色荧光（FITC）对红色荧光（PI）的双参数图中分别对未染色的活细胞、早期凋亡细胞和坏死细胞进行计数，以生成剂量反应曲线。
发现细胞活力的EC50值比集落试验的存活数据高1 - 4倍，类似于通过MTT或结晶紫活细胞染色获得的数据。通过膜联蛋白V结合测量的凋亡百分比与吖啶橙染色后凋亡细胞的显微镜评分一致。
所描述的FC测定法能很好地估计细胞活力，类似于活细胞染色试验的数据，并提供有关凋亡和坏死的额外信息。来自膜联蛋白V结合的FC数据与吖啶橙染色后的显微镜评分非常一致。
