用改良的MTT法测定rhG-CSF活性  

Improved MTT assay for determinating bioactivity of rhG-CSF

作　　者：周涛[1] 戴红梅[1] 汤国营[1] 孟庆东[1] 朱厚础[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071

出　　处：《生物技术通讯》1999年第4期279-283,共5页Letters in Biotechnology

摘　　要：ＭＴＴ测定法是根据线粒体脱氢酶催化ＭＴＴ形成蓝色甲■的多少来检测活细胞数和功能状态的，但原始方法中存在着一些问题，如敏感性偏低、有机溶剂产生蛋白质沉淀以及产物的溶解度偏低等。为了摸索测定 ｒｈＧ－ＣＳＦ活性的最适条件，我们以 ＮＦＳ－６０细胞为对象，比较了多种溶解缓冲液，并且对细胞数、ＭＴＴ浓度及保留时间、溶解液用量等条件进行了选择。结果表明，ＤＭＦ－２０％ＳＤＳ和 ２０％ＳＤＳ的效果最好，测定时细胞数为每孔 １０００个细胞，所加 ＭＴＴ浓度为 １ｍｇ／ｍｌ，保留时间为 ４ ｈ，溶解液的用量为每孔 １００μｌ。MTT assay is a convenient method to estimate the number of viable cells, which de- pends on the reduction of tetrazolium salt, MTT, to form a blue formanzan product by living cells. But there are several limitations in the original technique, which developed by Mosmann, such as low sensitivity, protein precipitation, and low solubility of the product. These problems have been overcome by some modifications in our assays used of NFS-60 cell line. So the optimal procedure for determinating the bioactivity of rhG-CSF has been established: the initial cell num- ber was 1 000 per well, MTT concentration was 1 mg/ml, incubation time was 4 hours, forman- zan solute buffer was 50% DMF-20% SDS or 0. 01 NHC-20% SDS, and solution volume was 100μl per well. With these modifications, MTT assay can replace the [3H] thymidine uptake as- say to measure cell proliferation or survival in growth factor.

关 键 词：MTT 甲臜 细胞活性 粒细胞集落刺激因子 

分 类 号：Q503[生物学—生物化学]