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

• Published in: Toxicology in vitro 2013-03, Vol.27 (2), p.954-963
• Main Authors: Aranda, A., Sequedo, L., Tolosa, L., Quintas, G., Burello, E., Castell, J.V., Gombau, L.
• Format: Article
• Language: English
• Subjects: Animals; Biological Assay - methods; Cell Survival - drug effects; Cells, Cultured; DCFH-DA; Ferric Compounds - toxicity; Ferrosoferric Oxide - toxicity; Fluoresceins; Fluorescent Dyes; Hepatocytes; In vitro assays; Lactic Acid - toxicity; Male; Nanoparticles; Nanoparticles - toxicity; Oxidative Stress - drug effects; Polyethylene Glycols - toxicity; Polyglycolic Acid - toxicity; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species - metabolism; Silicon Dioxide - toxicity; Titanium - toxicity
• ISSN: 0887-2333; 1879-3177
• DOI: 10.1016/j.tiv.2013.01.016

► Nanomaterials interfere with most in vitro fluorimetric screening tests. ► We explore the usefulness of DCFH-DA test to asses NP-induced oxidative stress. ► NP uptake correlated with quenching of dye fluorescence emission. ► Minor changes in DCFH-DA protocol allowed quantification of free radical formation. ► Protocol modifications did not result in free radical formation for all tested NPs. 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-dihydro-fluorescein 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 25nm and 50nm 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.