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Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay: A quantitative method for oxidative stress assessment of nanoparticle-treated cells

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Author: Aranda, A. · Sequedo, L. · Tolosa, L. · Quintas, G. · Burello, E. · Castell, J.V. · Gombau, L.
Source:Toxicology in Vitro, 2, 27, 954–963
Identifier: 525875
doi: doi:10.1016/j.tiv.2013.01.016
Keywords: Nutrition · DCFH-DA · Hepatocytes · In vitro assays · Nanoparticles · Food and Nutrition · Healthy Living · Life · RAPID - Risk Analysis for Products in Development · ELSS - Earth, Life and Social Sciences


No consensus exists on how to address possible toxicity of nanomaterials as they interfere with most in vitro screening tests based on colorimetric and fluorimetric probes such as the dichloro-dihydrofluorescein diacetate (DCFH-DA) assay for detection of oxidative species. In the present research, nanomaterial interaction with DCFH-DA was studied in relation to its nature and/or assay conditions (cell-based and time exposure) by incubating Rhodamine (Rhd)-labeled 25 nm and 50 nm silica (SiO2), naked and oleic acid coated magnetite, (Fe3O4) and maghemite (Fe2O3) iron oxide, titanium dioxide (TiO2) and poly(ethylene oxide)-poly(lactide/glycolide) acid (PLGA-PEO) nanoparticles (NPs) with metabolically active rat hepatocytes for 4 and 24-h periods. Data indicated that nanoparticle uptake correlated with quenching of dye fluorescence emission. In spite of their masking effect, the oxidative potential of NPs could be detected at a limited threshold concentration when exposed for periods of time longer than those frequently used for this test. However, changes in the experimental conditions did not systematically result in free radical formation for all nanomaterials tested. Overall data indicate that despite the quenching effect of nanoparticles on DCFH-DA assay, it can be considered as a useful tool for quantitative measurement of NPs-induced oxidative stress by minor modifications of standardized protocols.