A fluorescence microplate cytotoxicity assay with a 4-log dynamic range that identifies synergistic drug combinations
A fluorescence microplate cytotoxicity assay with a 4-log dynamic range that identifies synergistic drug combinations
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DOI：
10.1158/1535-7163.mct-04-0331
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发表时间：
2007-03-01
影响因子：
5.7
通讯作者：
Reynolds, C. Patrick
中科院分区：
文献类型：
--
作者：
Frgala, Tomas;Kalous, Ondrej;Reynolds, C. Patrick
目的：96孔组织培养板中的细胞毒性测定允许快速处理多条件实验的样品，但动态范围有限。使用DIMSCAN，一种用于定量组织培养板中相对细胞数量的荧光数字图像系统，我们开发了一种动态范围> 4-log的96孔细胞毒性测定法。研究方法：为了克服限制用荧光素二乙酸酯检测活细胞的背景荧光，我们使用2 '4' 5 '6'-四溴-荧光素（曙红Y）淬灭培养基和非活细胞中的背景荧光，以增强用数字图像阈值化实现的背景荧光的减少。用神经母细胞瘤和白血病细胞系的系列稀释液测试了新方法的灵敏度和线性。DIMSCAN与其他体外细胞毒性试验进行了比较：3-（4，5-二甲基噻唑-2-基）-2，5-二苯基四唑溴化物、菌落形成和台盼蓝染料排除。结果如下：在没有背景荧光减少的情况下，扫描在100至10（6）个细胞/孔的各种细胞浓度下产生几乎平坦的曲线。无论是数字图像阈值还是曙红Y都显著降低了背景荧光，并且将它们组合在一起，即使在每孔存在4 × 10（4）个非活细胞的情况下，在> 4个对数的动态范围内，相对荧光与活细胞数也实现了线性相关（r > 0.9）。通过DIMSCAN测定法测量的去铁胺对神经母细胞瘤细胞系的细胞毒性实现了与通过手动台盼蓝计数或软琼脂中的集落形成获得的数据相似的剂量-反应曲线，但具有更宽的动态范围。长期培养物记录了DIMSCAN在整个动态范围内检测到的活细胞的克隆形成能力。使用DIMSCAN和3-（4，5-二甲基噻唑-2-基）-2，5-二苯基溴化四唑（MTT）测定法测试两种药物组合（丁噻呋砜亚胺+美法仑或芬维A胺+沙芬戈）的细胞毒性，并且DIMSCAN的更宽的动态范围促进了协同相互作用的检测。结论：DIMSCAN能够在96孔板中快速有效地进行细胞毒性试验，动态范围> 4 log。该测定能够快速检测微孔板中的抗癌药物组合。
Purpose: Cytotoxicity assays in 96-well tissue culture plates allow rapid sample handling for multicondition experiments but have a limited dynamic range. Using DIMSCAN, a fluorescence digital image system for quantifying relative cell numbers in tissue culture plates, we have developed a 96-well cytotoxicity assay with a > 4-log dynamic range. Methods: To overcome background fluorescence that limits detection of viable cells with fluorescein diacetate, we used 2'4'5'6'-tetrabromo-fluorescein (eosin Y) to quench background fluorescence in the medium and in nonviable cells to enhance the reduction of background fluorescence achieved with digital image thresholding. The sensitivity and linearity of the new assay were tested with serial dilutions of neuroblastoma and leukemia cell lines. DIMSCAN was compared with other in vitro cytotoxicity assays: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation, and trypan blue dye exclusion. Results: Without background fluorescence reduction, scans produced a nearly flat curve across various cell concentrations from 100 to 10(6) cells per well. Either digital image thresholding or eosin Y dramatically reduced background fluorescence, and combining them achieved a linear correlation (r > 0.9) of relative fluorescence to viable cell number over > 4 logs of dynamic range, even in the presence of 4 x 10(4) nonviable cells per well. Cytotoxicity of deferoxamine for neuroblastoma cell lines measured by the DIMSCAN assay achieved dose-response curves similar to data obtained by manual trypan blue counts or colony formation in soft agar but with a wider dynamic range. Long-term cultures documented the clonogenic ability of viable cells detected by DIMSCAN over the entire dynamic range. The cytotoxicity of two drug combinations (buthionine sulfoximine + melphalan or fenretinide + safingol) was tested using both DIMSCAN and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and the wider dynamic range of DIMSCAN facilitated detection of synergistic interactions. Conclusion: DIMSCAN offers the ability to rapidly and efficiently conduct cytotoxicity assays in 96-well plates with a dynamic range of > 4 logs. This assay enables rapid testing of anticancer drug combinations in microplates.