Pitfalls and limitations in using 4,5-diaminofluorescein for evaluating the influence of polyphenols on nitric oxide release from endothelial cells

Free Radical Biology & Medicine
Klaus Uhlenhut, Petra Högger

Abstract

The reagent 4,5-diaminofluorescein (DAF-2) is a widely utilized and sensitive fluorescent probe for real-time assessment of nitric oxide (NO) production. In this study we investigated the feasibility of using DAF-2 for detection of NO release from EA.hy 926 human endothelial cells stimulated with plant polyphenols. Flavonoids have recently gained much interest because of reported beneficial effects on vasodilatation, which have been ascribed to stimulation of endothelial NO production. DAF-2 shows moderate fluorescence, and because certain phenolic compounds quench fluorescence or fluoresce themselves, we utilized liquid chromatography to avoid interference. Our investigations with (+)-catechin and trans-resveratrol as test phenolic compounds revealed various previously undescribed principal methodologic pitfalls and limitations. Under assay conditions (+)-catechin displayed a highly significant increase in fluorescence intensity so that a control of test compound stability is advisable. Moreover, DAF-2 was subject to conversion to triazolofluorescein (DAF-2T) under certain assay and storage conditions; thus control of spontaneous reagent conversion is advisable. Finally, formation of DAF-2T was dose-dependently inhibited by po...Continue Reading

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Citations

Dec 8, 2015·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Namrata Singh, Debasish Bhattacharyya
Sep 25, 2012·Nitric Oxide : Biology and Chemistry·Anke BöhmerDimitrios Tsikas
Dec 25, 2015·Circulation Research·Andreas M BeyerDavid D Gutterman
May 4, 2021·The Ocular Surface·Shyam Sunder TummanapalliMark D P Willcox
Jul 30, 2021·Nitric Oxide : Biology and Chemistry·Junjie LiMiriam M Cortese-Krott

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