title: The reduction of water-soluble tetrazolium salt reagent on the plasma membrane of epidermal keratinocytes is oxygen dependent.
abstract: The water-soluble tetrazolium salt (WST-1) assay is frequently used to assess cell proliferation. However, our study showed that in normal and cancerous keratinocytes, this assay is more responsive to changes in oxygenation than to rates of cell growth. Stimulation of keratinocyte proliferation by low Ca(2+) and suppression of proliferation by nocodazole resulted in modest changes in WST-1 readings, whereas gradually reducing the level of oxygen in the cellular environment from ambient (21%) to near anoxic (0.1%) revealed a very strong negative correlation between cell oxygenation and WST-1 reagent reduction. In contrast, the very similar MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay, which uses a different tetrazolium salt, showed no sensitivity to the level of oxygen. Unlike MTT, WST-1 reagent is reduced extracellularly through trans-plasma membrane transport (tPMET), thereby suggesting that tPMET is oxygen dependent. We propose that the WST-1 assay can be developed into a sensitive quantitative method to evaluate cell oxygenation in vitro and used to study the role of hypoxia and tPMET in homeostasis and disease (e.g., cancer). At the same time, WST-1 assay should be used cautiously to assess cell viability or proliferation because readings can be affected by certain extrinsic (low atmospheric oxygen or high density culture) or intrinsic (defects in oxygen-sensing pathways) factors.
authors.affiliation: Centre for Oncology and Molecular Medicine, Division of Medical Sciences, College of Medicine, Dentistry, & Nursing, Ninewells Hospital, University of Dundee, UK.
authors.forename: Lynda
authors.initials: L
authors.lastname: Weir
authors.forename: Douglas
authors.initials: D
authors.lastname: Robertson
authors.forename: Irene M
authors.initials: IM
authors.lastname: Leigh
authors.forename: Andrey A
authors.initials: AA
authors.lastname: Panteleyev
mesh_terms: D000818:Animals; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D015739:Nocodazole / Q000494:pharmacology; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D013778:Tetrazolium Salts / Q000378:metabolism*; D050257:Tubulin Modulators / Q000494:pharmacology
chemical_list: C110656:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; D007202:Indicators and Reagents; D013778:Tetrazolium Salts; D050257:Tubulin Modulators; D010100:Oxygen; D015739:Nocodazole