A new fluorometric assay for cytotoxicity measurements in-vitro.
A new fluorometric assay for cytotoxicity measurements in-vitro.
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DOI：
10.3892/ijo.3.3.473
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发表时间：
1993-09
影响因子：
5.2
通讯作者：
B. Pagé;M. Pagé;C. Noel
中科院分区：
文献类型：
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作者：
B. Pagé;M. Pagé;C. Noel
抗癌药物的细胞毒性测试需要灵敏、可重复且适用于使用自动化仪器进行大规模测试的技术。目前，这些测定是通过使用染料、活力染色剂或依赖于底物的能量（例如 MTT 或 XTT）对细胞蛋白进行终点染色来进行的。虽然可靠。这些测定不够灵敏，对于大规模筛选来说太昂贵，或者它们使用的试剂可能对人员或设备有害。我们描述了使用阿拉玛蓝（Alamar Blue），一种新型非荧光底物，在活细胞还原后，产生非常强的荧光产物。使用自动荧光板读取器 Cytoflor，我们评估了各种参数，例如底物浓度、孵育时间和体积的线性度和检测下限。我们发现，对于两小时的测定，这种新型无毒底物可以检测每孔低至 200 个细胞，有效测量范围高达每孔 20,000 个细胞。荧光测定的灵敏度是比色测定的十倍以上。当用该测定法测量柔红霉素的细胞毒性并与 XTT 甲臜测定法进行比较时，我们发现了可比的 IC50 值，但这种新测定法更经济，并且与甲臜测定法相比，在两小时内即可获得结果。这种新的经济且多功能的检测方法可有利地用于抗癌药物和其他细胞毒性药物的大规模体外筛选。
Cytotoxicity testing of anticancer drugs requires techniques which are sensitive, reproducible and applicable to large scale testing using automated instruments. These assays are presently performed with end point staining of cell proteins with dyes, viability stains or energy dependent of substrates such as MTT or XTT. Although reliable. these assays are not sensitive enough, too expensive for large scale screening or they use reagents that may be harmful for personnel or equipment. We describe, the use of Alamar Blue, a new non fluorescent substrate, which after reduction in living cells, yields a very strong fluorescent product. Using the automated fluorescence plate reader Cytofluor, we have evaluated the various parameters such as substrate concentration, time and volume of incubation with respect to linearity and lower limit of detection. We found that for a two hour assay, this new non toxic substrate could detect as low as 200 cells per well with a useful measurement range up to 20,000 cells per well. The fluorescent assay is more than ten times as sensitive as the colorimetric assay. When the cytotoxicity of daunorubicin was measured with this assay and compared to the XTT formazan assay we found comparable IC50 values but this new assay was more economical and results are obtained in two hours as compared to four hours for the formazan assay. This new economical and versatile assay could be used with advantage for large scale in vitro screening of anticancer drugs and other cytotoxic agents.