Estimation of cell survival by flow cytometric quantification of fluorescein diacetate/propidium iodide viable cell number.
Estimation of cell survival by flow cytometric quantification of fluorescein diacetate/propidium iodide viable cell number.
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发表时间：
1989-07
期刊：
影响因子：
11.2
通讯作者：
Douglas D. Ross;Christopher C. Joneckis;José V. Ordóñez;Allison M. Sisk;Richard K. Wu;Anne W. Hamburger;Richard E. Nora
中科院分区：
文献类型：
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作者：
Douglas D. Ross;Christopher C. Joneckis;José V. Ordóñez;Allison M. Sisk;Richard K. Wu;Anne W. Hamburger;Richard E. Nora
我们报告了一种流式细胞术的方法来量化的存活细胞的数量仍然在悬浮培养后暴露于细胞毒性药物。在用荧光素二乙酸酯和碘化丙啶在等渗溶液中染色后，通过流式细胞术测量细胞荧光来评估细胞活力。通过活细胞的定时计数和流式细胞仪样品流速的知识确定每ml培养物中的活细胞数。使用培养的P388鼠或HL-60人白血病细胞作为模型系统。该方法可以准确地定量悬浮培养物中的活细胞浓度，从100个细胞/ml到1百万个细胞/ml。如果在短暂（1-4小时）暴露于药物后，在流式细胞术计数活细胞之前允许更长时间的细胞死亡和溶解，则该方法作为细胞毒性试验的灵敏度增加。如果在药物（柔红霉素、放线菌素D、长春新碱）暴露后将活力评估延迟至少72 h，则获得的结果与软琼脂克隆形成试验或比色3-（4，5-二甲基噻唑-2-基）-2，5-二苯基四唑溴化物试验获得的结果接近。在研究长春新碱、放线菌素D、1-β-D-阿拉伯呋喃糖基胞嘧啶和柔红霉素对对这些药物敏感和耐药的P388或HL-60细胞的细胞毒性作用时，通过流式细胞术计数活细胞数获得了合理的结果。我们已经能够使用从急性髓性白血病患者获得的骨髓母细胞轻松地进行流式细胞术活力测定。该方法简便，相对快速，并且由于它是研究悬浮培养细胞的理想方法，因此其作为白血病化疗反应预测因子的潜力值得进一步评估。
We report a flow cytometric method to quantify the number of viable cells remaining in suspension culture following exposure to cytotoxic drugs. Cell viability is assessed by flow cytometric measurement of cellular fluorescence after staining with fluorescein diacetate and propidium iodide in isotonic solution. The number of viable cells per ml of culture is determined by a timed count of viable cells and from knowledge of the flow cytometer sample flow rate. P388 murine or HL-60 human leukemia cells in culture were used as model systems. This method can quantify accurately viable cell concentrations in suspension culture from 100 cells/ml to 1 million cells/ml. The sensitivity of the method as a cytotoxicity assay increases if, following brief (1-4-h) exposure to drug, greater time is allowed for cell death and lysis to occur prior to flow cytometric counting of viable cells. If the viability assessment is deferred for at least 72 h following drug (daunorubicin, actinomycin D, vincristine) exposure, results were obtained approximating those obtained from the soft agar clonogenic assay or the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In studying the cytotoxic effects of vincristine, actinomycin D, 1-beta-D-arabinofuranosylcytosine, and daunorubicin on P388 or HL-60 cells sensitive and resistant to these agents, reasonable results were obtained by flow cytometric counting of viable cell number. We have been able to perform this flow cytometric viability assay with ease using bone marrow blast cells obtained from patients with acute myelogenous leukemia. The method is facile, relatively rapid, and since it is ideal for studying cells in suspension culture, its potential as a predictor of chemotherapeutic response in leukemia warrants further evaluation.