RAPID COLORIMETRIC ASSAY FOR CELL-GROWTH AND SURVIVAL - MODIFICATIONS TO THE TETRAZOLIUM DYE PROCEDURE GIVING IMPROVED SENSITIVITY AND RELIABILITY
RAPID COLORIMETRIC ASSAY FOR CELL-GROWTH AND SURVIVAL - MODIFICATIONS TO THE TETRAZOLIUM DYE PROCEDURE GIVING IMPROVED SENSITIVITY AND RELIABILITY
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DOI：
10.1016/0022-1759(86)90368-6
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发表时间：
1986-01-01
影响因子：
2.2
通讯作者：
LANG, R
中科院分区：
文献类型：
--
作者：
DENIZOT, F;LANG, R
估计在微滴盘孔中生长的活细胞数量的一种方便方法是使用比色法和自动微滴板扫描分光光度计。莫斯曼发明了一种这样的测定方法，它依赖于活细胞对四氮唑盐（MTT）的还原，以形成蓝色的甲酸产物。然而，原始技术有几个技术限制，即灵敏度低于最佳值，由于添加有机溶剂溶解蓝色甲醛产物时蛋白质沉淀而产生的可变本底，以及产品的溶解度低。这些问题已经通过以下修改得以克服：避免在培养培养基中使用血清，从而克服了有机溶剂中的沉淀问题；避免在培养介质中使用酚红，从而避免在最终溶剂中使用酸，从而改变甲酸的光谱特性；反应后去除含MTT的培养基，随后用纯丙醇或乙醇快速溶解甲酰胺；使用高浓度的MTT；使用半面积微滴盘，以增加从给定量的甲醛分光光度计读数；在双波长分光光度计中使用更明智的参考波长。通过这些改进，该试验的可靠性和灵敏度已经提高到在许多情况下可以取代[3H]胸苷摄取试验来测量生长因子或细胞毒性试验中的细胞增殖或存活。给出了其在IL-2测定中的应用实例。
A convenient way to estimate the number of viable cells growing in microtitre tray wells is to use a colorimetric assay and an automatic microplate scanning spectrophotometer. One such assay, developed by Mosmann, depends on the reduction by living cells of tetrazolium salt, MTT, to form a blue formazan product. However the original technique has several technical limitations, namely a less than optimal sensitivity, a variable background due to protein precipitation on adding an organic solvent to dissolve the blue formazan product, and a low solubility of the product. These problems have been overcome by the following modifications: avoidance of serum in the incubation medium, thus overcoming precipitation problems in the organic solvent; avoidance of phenol red in the incubation medium, thus avoiding the use of acid in the final solvent which altered the spectral properties of the formazan; elimination of the medium containing MTT after the reaction and subsequent use of pure propanol or ethanol to rapidly solubilize the formazan; use of a higher concentration of MTT; use of half-area microtitre trays to increase the spectrophotometer readings from a given amount of formazan; use of a more judicious reference wavelength in a dual wavelength spectrophotometer. With these modifications the reliability and sensitivity of the test have been increased to the point where it can in many cases replace the [3H]thymidine uptake assay to measure cell proliferation or survival in growth factor or cytotoxicity assays. Examples of its use in IL-2 assays are given.