Superoxide produced by activated neutrophils efficiently reduces the tetrazolium salt, WST-1 to produce a soluble formazan: A simple colorimetric assay for measuring respiratory burst activation and for screening anti-inflammatory agents
Abstract
Activation of the respiratory burst of granulocytes and macrophages by invading microorganisms is a key first line cellular defence against infection. Failure to generate this response leads to persistent life-threatening infection unless appropriate antibiotic treatment is given. The respiratory burst of neutrophils is usually measured spectrophotometrically by following ferricytochrome c reduction, and histologically by using the tetrazolium salt, nitroblue tetrazolium, which is reduced intracellularly to an insoluble formazan. In both assays, reduction is mediated by superoxide generated via NADPH oxidase. Because ferricytochrome c has a high molecular mass and high background absorbance at 550 nm, the assay lacks sensitivity and is not ideally suited to microplate measurement. We have circumvented these limitations by using the cell-impermeable, sulfonated tetrazolium salt, WST-1, which exhibits very low background absorbance and is efficiently reduced by superoxide to a stable water-soluble formazan with high molar absorptivity. This has permitted adaptation of the WST-1 assay to microplate format while retaining sensitivity. Reduction of WST-1 by activated human peripheral blood neutrophils correlated closely with ferricytochrome c reduction across a range of PMA concentrations and with time of activation by PMA and fMLP. Reduction of WST-1 was inhibited by 98% by superoxide dismutase (20 microg/ml) and by 88% by the NADPH oxidase inhibitor, diphenyleneiodinium (10 microM) but was resistant to catalase, azide and the NADH oxidase inhibitor, resiniferatoxin. WST-1 and ferricytochrome c reduction were also compared using xanthine/xanthine oxidase to generate superoxide. Under optimised assay conditions, both WST-1 and ferricytochrome c reduction were directly proportional to added xanthine. WST-1 generated approximately 2-fold greater increase in absorbance than ferricytochrome c at their respective wavelengths, and this translated into increased assay sensitivity. Addition of the intermediate electron acceptor, 1-methoxy phenazine methosulfate, increased the background of the neutrophil assay but did not affect the overall magnitude of the response. We have used the WST-1 assay to assess human neutrophil dysfunction and to compare anti-inflammatory activity.
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WST-1, a cell-impermeable sulphonated tetrazolium salt, has very low background absorbance and is efficiently reduced by superoxide to a stable water-soluble formazan with high molar absorptivity.
“To overcome this limitation, the cell-impermeable sulphonated tetrazolium salt, WST-1, which exhibits a very low background absorbance and is efficiently reduced by superoxide to a stable, water-soluble formazan with high molar absorptivity, is used [154,155], Figure 61.”
From Superoxide Anion Chemistry—Its Role at the Core of the Innate Immunity (2023) · cites this paper as [155]
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- Authors
- Tan, A. S., & Berridge, M. V.
- Affiliations
- Malaghan Institute of Medical Research (NZ)
- Corresponding author
- Berridge, Michael V
- Venue
- Journal of Immunological Methods
- Publisher
- Elsevier BV
- Published
- 2000-04
- Volume
- 238
- Issue
- 1-2
- Pages
- 59-68
- ISSN
- 0022-1759 (ISSN-L)
- OpenAlex
- W2054789114
- Cited by
- 330
- References
- 29
- Citations by year
- 2026: 4 · 2025: 8 · 2024: 6 · 2023: 13 · 2022: 8 · 2021: 14 · 2020: 8 · 2019: 12 · 2018: 9 · 2017: 12 · 2016: 19 · 2015: 18 · 2014: 23 · 2013: 16 · 2012: 14
- FWCI
- 2.01
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Immunological Methods (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Immunology and Microbiology › Immunology › Neutrophil, Myeloperoxidase and Oxidative Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Toxicology › Bioactive Compounds and Antitumor Agents
- Keywords
- neutrophil respiratory burst, superoxide, formazan, NADPH oxidase, neutrophil activation, ferricytochrome c reduction, anti-inflammatory agents, neutrophil dysfunction, microplate assay, superoxide dismutase, xanthine/xanthine oxidase
- MeSH terms
- Anti-Inflammatory Agents, Cells, Cultured, Colorimetry, Cytochrome c Group, Drug Evaluation, Preclinical, Formazans, Humans, Multienzyme Complexes, NADH, NADPH Oxidoreductases, Neutrophils, Superoxides, Superoxide Dismutase, Tetrazolium Salts, Respiratory Burst, Neutrophil Activation, NADPH Oxidases
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