The use of intermediate electron acceptors to enhance MTT bioreduction in a microculture tetrazolium assay for human growth hormone.
We contrast the effects of three intermediate electron acceptors (IEAs) on the highly quantitative ESTA bioassay for human growth hormone. This is a microculture tetrazolium assay based upon the in vitro reduction of the tetrazolium salt MTT, by Nb2 cells which have been activated with hGH. Each of the IEAs influenced MTT-formazan production in a distinctive manner. The two quinonoids, namely menadione and co-enzyme Q0 markedly increased the MTT-formazan produced by hormone activated Nb2 cells and thereby amplified the response of our bioassay for human growth hormone (hGH). The exceptionally low bioassay baseline which is characteristic of the unstimulated Nb2 cells when only MTT is added was retained in the presence of CoQ0, but was greatly increased by menadione. Phenazine methosulphate, which is the most widely used redox intermediary in microculture tetrazolium assays, also increased the baseline, but had only a minimal additional effect on MTT reduction by activated Nb2 cells. We conclude that CoQ0 is the preferred IEA for this ESTA bioassay for hGH.
Goodwin
C J
CJ
Department of Molecular Pathology, University College London, UK.
Holt
S J
SJ
Downes
S
S
Marshall
N J
NJ
D000818:Animals; D004583:Electrons; D013006:Growth Hormone / Q000032:analysis*; D006801:Humans; D010084:Oxidation-Reduction; D051381:Rats; D013778:Tetrazolium Salts / Q000737:chemistry*; D013844:Thiazoles / Q000737:chemistry*; D014407:Tumor Cells, Cultured
D013778:Tetrazolium Salts; D013844:Thiazoles; D013006:Growth Hormone; C022616:thiazolyl blue