A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1)

Peskin, Alexander V.; Winterbourn, Christine C.

doi:10.1016/s0009-8981(99)00246-6

Cited by 451 publications

(269 citation statements)

References 14 publications

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“…produced was determined by measuring the superoxide dismutase-inhibitable reduction of tetrazolium salt, WST-1 (35)(36)(37). Primary microglia were plated at 5 ϫ 10 4 /well in 200 l of culture medium in 96-well plates and incubated for 24 h at 37°C in a humidified atmosphere of 5% CO 2 and 95% air.…”

Section: Methodsmentioning

confidence: 99%

NADPH Oxidase Mediates Lipopolysaccharide-induced Neurotoxicity and Proinflammatory Gene Expression in Activated Microglia

Qin

Liu

Wang

et al. 2004

Journal of Biological Chemistry

539

492

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Parkinson's disease is characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra. We have previously reported that lipopolysaccharide (LPS)-induced degeneration of dopaminergic neurons is mediated by the release of proinflammatory factors from activated microglia. Here, we report the pivotal role of NADPH oxidase in inflammation-mediated neurotoxicity, where the LPS-induced loss of nigral dopaminergic neurons in vivo was significantly less pronounced in NADPH oxidase-deficient (PHOX

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Section: Methodsmentioning

confidence: 99%

NADPH Oxidase Mediates Lipopolysaccharide-induced Neurotoxicity and Proinflammatory Gene Expression in Activated Microglia

Qin

Liu

Wang

et al. 2004

Journal of Biological Chemistry

539

492

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“…Superoxide dismutase (SOD) activity was measured, using the method described by Peskin and Winterbourn [32], by monitoring the hypoxanthine and xanthine oxidase-generated superoxide-dependent reduction of the tetrazolium dye, WST-1 ((2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt; Dojindo Molecular Technologies). In brief, assay buffer (50 mM sodium phosphate (pH 8.0) containing 0.1 mM DTPA (diethylenetriaminepentaacetic acid; Sigma-Aldrich), 0.1 mM hypoxanthine (Sigma-Aldrich) and 50 µM WST-1) was added to each well of a 96 well plate containing 100 µg of sample protein.…”

Section: Superoxide Dismutase Activitymentioning

confidence: 99%

Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia

Ferguson

Smerdon

Harries

et al. 2018

Free Radical Biology and Medicine

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In vivo, mammalian cells reside in an environment of 0.5-10% O 2 (depending on the tissue location within the body), whilst standard in vitro cell culture is carried out under room air. Little is known about the effects of this hyperoxic environment on treatment-induced oxidative stress, relative to a physiological oxygen environment. In the present study we investigated the effects of long-term culture under hyperoxia (air) on photodynamic treatment. Upon photodynamic irradiation, cells which had been cultured long-term under hyperoxia generated higher concentrations of mitochondrial reactive oxygen species, compared with cells in a physioxic (2% O 2 ) environment. However, there was no significant difference in viability between hyperoxic and physioxic cells. The expression of genes encoding key redox homeostasis proteins and the activity of key antioxidant enzymes was significantly higher after the long-term culture of hyperoxic cells compared with physioxic cells. The induction of antioxidant genes and increased antioxidant enzyme activity appear to contribute to the development of a phenotype that is resistant to oxidative stress-induced cellular damage and death when using standard cell culture conditions. The results from experiments using selective inhibitors suggested that the thioredoxin antioxidant system contributes to this phenotype. To avoid artefactual results, in vitro cellular responses should be studied in mammalian cells that have been cultured under physioxia. This investigation provides new insights into the effects of physioxic cell culture on a model of a clinically relevant photodynamic treatment and the associated cellular pathways.

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“…Total SOD activity was assessed by the disappearance of superoxide detected by a tetrazolium salt. 24 Catalase activity was determined as the conversion of methanol to formaldehyde in the presence of H 2 O 2 using the method of Johansson and Borg. 25 GPx activity was measured indirectly by measuring the decrease in NADPH absorbance at 340 nm, because it is converted to oxidized nicotinamide-adenine dinucleotide phosphate by glutathione reductase.…”

Section: Total Sod Catalase and Gpx Activitiesmentioning

confidence: 99%

NADPH Oxidase in the Renal Medulla Causes Oxidative Stress and Contributes to Salt-Sensitive Hypertension in Dahl S Rats

et al. 2006

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Abstract-Dahl salt-sensitive (SS) rats exhibit increased renal medullary oxidative stress and blood pressure salt-sensitivity compared with consomic, salt-resistant SS-13 BN rats, despite highly similar genetic backgrounds. The present study examined potential sources of renal medullary superoxide in prehypertensive SS rats fed a 0.4% NaCl diet by assessing activity and protein levels of superoxide producing and scavenging enzymes. Superoxide production was nearly doubled in SS rats compared with SS-13 BN rats as determined by urinary 8-isoprostane excretion and renal medullary oxy-ethidium microdialysate levels. Medullary superoxide production in tissue homogenates was greater in SS rats, and the NADPH oxidase inhibitor diphenylene iodonium preferentially reduced SS levels to those found in SS-13 BN rats. Dinitrophenol, a mitochondrial uncoupler, eliminated the remaining superoxide production in both strains, whereas inhibition of xanthine oxidase, NO synthase, and cycloxygenase had no effect. L-arginine, NO synthase, superoxide dismutase, catalase, and glutathione peroxidase activities between SS and SS-13 BN rats did not differ. Chronic blood pressure responses to a 4% NaCl diet were then determined in the presence or absence of the NADPH oxidase inhibitor apocynin (3.5 g/kg per minute), chronically delivered directly into the renal medulla. Apocynin infusion reduced renal medullary interstitial superoxide from 1059Ϯ130 to 422Ϯ80 (oxyethidium fluorescence units) and mean arterial pressure from 175Ϯ4 to 157Ϯ6 mm Hg in SS rats, whereas no effects on either were observed in the SS-13 BN . We conclude that excess renal medullary superoxide production in SS rats contributes to salt-induced hypertension, and NADPH oxidase is the major source of the excess superoxide.

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A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1)

Cited by 451 publications

References 14 publications

NADPH Oxidase Mediates Lipopolysaccharide-induced Neurotoxicity and Proinflammatory Gene Expression in Activated Microglia

NADPH Oxidase Mediates Lipopolysaccharide-induced Neurotoxicity and Proinflammatory Gene Expression in Activated Microglia

Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia

NADPH Oxidase in the Renal Medulla Causes Oxidative Stress and Contributes to Salt-Sensitive Hypertension in Dahl S Rats

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