MTT显色反应实验条件分析

Examination and analyses to the coloring reacting of MTT

目的 :检验分析MTT实验方法本底高、灵敏度低、稳定性差的影响因素 ,改进实验条件提高灵敏度和稳定性 ,降低本底。方法 :用MTT比色方法 ,细胞毒试验及细胞增殖实验 ,分析比较不同条件下MTT显色反应的性能特点。结果 :比较了 13种溶剂对Formazan的溶解性能。分析检验了琥珀酸脱氢酶在细胞上的分布特点、pH值对MTT反应体系的影响、酚红和牛血清对检验结果的影响 ,提出相应对策。检测了MTT反应时间曲线和细胞活力曲线。分析了改进后的MTT实验方法的灵敏度和稳定性。结论 :以MTT为底物的酶在细胞代谢中主要分布在细胞膜表面。MTT反应体系的pH值为 6 7,比较好的Formazan溶剂是正丙醇、异丙醇和乙二醇乙醚。去掉细胞培养上清 ,检测细胞上的酶 ,用溶剂使蛋白质变性 ,经沉淀去掉蛋白质影响 ,能使MTT实验方法的本底降低 ,灵敏度提高。通常情况下该方法检测K 5 62细胞和人淋巴细胞的灵敏度分别为 10 0 0和 10 0 0 0个细胞左右。

Objective: To improve experimental conditions of the colorimetric assay,depends on the reduction by living cells of tetrazolium salt,MTT,and to enhance sensitivity and reliability and to lower background.Methods: The colorimetric assay with MTT,and the experiments of cytotoxicity and proliferation of cells.Results: The analyses and examining to the experimental conditions of the colorimetric assay with MTT have found that majority of the mitochondrial enzyme succinate dehydrogenase is distributed over surface of cell membrane;the envirornment of reaction of enzyme with MTT is pH 6.7;following cells incubated in the common medium containing serums and phenol red were completed,the supernatant is discarded,and leaving cells alone react with MTT,and subsequent use of pure propanol to rapidly dissolve the formazan.The pure organic solvent can precipitate trace proteins to be removed by centrifugation and lower acid organic solvent (pH5.8) can also eliminate to the influence of phenol red to the assay.Conclusion: When the experimental conditions have be improved,the cure of cell enzyme reaction with MTT and the cure of MTT cleaved reaction time under in saturated state are linear relationship.The background is very lowed down;the sensitivity and reliability are heightened;the minimum detectable number of K 562 and Human T cells are respectively 1000 cells and 10000 cells in our hands.