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Tetrazolium Bioreduction Assays in Intact Cells: Requirements for Both Hexokinase (HK) and Glucose (Glc)
Author(s) -
Robey R. Brooks
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a812-d
Subject(s) - formazan , metabolism , hexokinase , biochemistry , chemistry , cell culture , biology , glycolysis , genetics
Tetrazolium bioreduction is widely used in cell proliferation and toxicity assays, but its molecular basis is incompletely defined in intact cells. Originally attributed to redox coupling with mitochondrial metabolism, recent evidence suggests coupling to extra mitochondrial metabolism and a specific requirement for Glc. To better understand the role of Glc and the first committed step of its metabolism (i.e. phosphorylation by HK) in this process, we examined both wild‐type (K1‐2) and HK‐deficient (MI5‐4) CHO cells for the ability to reduce a soluble tetrazolium salt (MTS). Cells were examined at confluence under basal growth conditions in both the presence and absence of Glc, but without confounding stresses or stimuli. Formazan chromophore formation by parental K1‐2 cells increased nine‐fold within 1 h in the presence, but not the absence, of Glc. In contrast, MTS reduction by HK‐deficient MI5‐4 cells was markedly impaired in the presence of Glc, an effect that was reversed by ectopic HK expression. Pyruvate or glutamine provision as alternative energy substrates, either individually or in combination, failed to mimic the effects of Glc. MTS reduction by cultured mesangial cells was similarly Glc‐dependent and was indistinguishable from that observed in K1‐2 cells. The rapidity and magnitude of these changes are incompatible with major corresponding proliferative effects or viability changes. They are consistent, however, with a major role for extramitochondrial metabolism involving Glc phosphorylation in tetrazolium bioreduction. These findings suggest the need for caution when interpreting tetrazolium‐based proliferation or toxicity assays under conditions that may directly influence metabolism (e.g. following growth factor stimulation) or where Glc concentrations are not known (e.g. following prolonged incubation).