MTT法检测rhEGF生物活性

目的鉴于待测的生物活性因子均有适于其自身的最佳测定条件,规范了重组表皮生长因子(rhEGF)的MTT测定法。方法根据MTT测定法原理,在3个因素(每孔细胞数、MTT和胎牛血清浓度)及3个水平上进行了正交实验设计。结果MTT检测法的最佳条件是:Balb/c3T3细胞悬液(5×107cells·L-1)100μl·well-1,MTT(8g·L-1)20μl·well-1,FBS(1·5%)100μl·well-1。结论上述正交实验规范的MTT最佳实验条件,适用于Balb/c3T3细胞活性的检测及其药物抑制性实验。但在测定rhEGF生物活性时,为使对照孔细胞数保持在较低水平以便给生物活性因子留有适当的余地以显示其促细胞增殖活性,建议FBS浓度采用0·5%。

Detection of rhEGF bioactivity by MTT assay

Aim Because the activity of a given bioactive factor to be detected has its own optimal conditions for detection,the MTT method for rhEGF assay was defined in this paper. Methods According to the principle of MTT assay,an orthogenal test was designed with three factors (cell per well,MTT and fetal bivine serum concentrations) at three destinct levels.Results The optinum conditions for MTT assay were as follows:Balb/c 3T3 cell suspension (5×107 cell·L-1) 100 μl·well-1,fetal bovine serum(1.5%)100 μl·well-1.MTT (8 g·L-1) 20 μl·well-1.Conclusions The above-mentioned optimal conditions for MTT assay defined by the orthogona ltest is suitable for the detection of Balb/c 3T3 cell bioactivity and its druginhibition test.However in the case of rhEGF assay,the cell counts of the control have to keep at a lower level in order to leave appropriate leeway to show the proliferation activity of the bioactive factor,thus a concentration of 0.5% ofFBS is recommended while rhEGF activity is detected.