The reduction of water-soluble tetrazolium salt reagent on the plasma membrane of epidermal keratinocytes is oxygen dependent

Lynda Weir, Douglas Robertson, Irene M. Leigh, Andrey A. Panteleyev

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    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 Ca2+ 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. (C) 2011 Elsevier Inc. All rights reserved.

    Original languageEnglish
    Pages (from-to)31-37
    Number of pages7
    JournalAnalytical Biochemistry
    Volume414
    Issue number1
    DOIs
    Publication statusPublished - 1 Jul 2011

    Keywords

    • Hypoxia
    • Keratinocyte
    • MTT assay
    • Proliferation
    • Trans-plasma membrane electron transport (tPMET)
    • WST-1 assay
    • ELECTRON-TRANSPORT
    • CELL-SURFACE
    • MAMMALIAN-CELLS
    • UP-REGULATION
    • HUMAN SKIN
    • HYPOXIA
    • GROWTH
    • NADH
    • DIFFERENTIATION
    • CONSUMPTION

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