[Improved MTT colorimetric assay for serum TNF activity].
The activity of serum tumor necrosis factor(TNF) is known to be related with the mechanism and prognosis of many diseases. The aim of this experiment was to look for an economic and reliable method for assaying the activity of serum TNF. We used the neutrophil solventor 20% SDS-50% DMF instead of the acidity solventor 0.04 mol/l acidified isopropy alcohol and established an improved 3-(4,5-dimethyl thiazoly)2,5-diphenyl-tetrazolium bromide (MTT) colorimetric assay for measuring the amount and function of living cell. We used this improved MTT colorimetric assay for measuring TNF activity of peripheral blood serum of healthy persons. It avoided the deposition of the protein in serum and medium and showed more repeatability, as compared with the conventional MTT colorimetric assay. The results showed: when the activity of target cell(TC)-L929 is > or = 95%, the density of TC is > or = 1 x 10(2)/well and the number of living cells in each well is positively correlated with the OD volume (OD570nm-OD630nm of purple formazan metabolite of MTT solution(r = 0.87, P < 0.01); when the density of TC is 5 x 10(4)/well, the level standard of TNF in each well is negatively correlated with the OD volume (OD570nm-OD630nm)r = 0.79, P < 0.01). Using this method, we measured the TNF activity of 20 healthy persons' peripheral blood serum. The mean +/- s of TNF activity is 20.95 +/- 3.2 IU/ml. This method is dependable, easy-to-do and economic, it has good repeatability within 3-12 hours.
Zhang
P
P
Department of Immunology, School of Basic Medical Sciences, Chengdu.
Zhang
C
C
Wei
D
D
Zhao
Z
Z
Liu
J
J
Cai
M
M
D000818:Animals; D003124:Colorimetry / Q000379:methods; D006801:Humans; D007739:L Cells; D051379:Mice; D013778:Tetrazolium Salts; D013844:Thiazoles; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis* / Q000378:metabolism
D013778:Tetrazolium Salts; D013844:Thiazoles; D014409:Tumor Necrosis Factor-alpha; C022616:thiazolyl blue