Real time monitoring and quantification of reactive oxygen species in breast cancer cell line MCF-7 by 2′,7′–dichlorofluorescin diacetate (DCFDA) assay

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dc.contributor.author Figueroa Gonzalez, Daniela
dc.contributor.author Asaduzzaman, Mohammad
dc.contributor.author Young, Fiona Margaret
dc.date.accessioned 2018-06-14T00:56:28Z
dc.date.available 2018-06-14T00:56:28Z
dc.date.issued 2018-04-07
dc.description Published by Elsevier Inc. This manuscript version is made available under the CC-BY-NC-ND 4.0 license: http://creativecommons.org/licenses/by-nc-nd/4.0/ This author accepted manuscript is made available following 12 month embargo from date of publication (April 2018) in accordance with the publisher’s archiving policy en_US
dc.description.abstract The detection of reactive oxygen species (ROS) using 2′,7′–dichlorofluorescin diacetate (DCFDA) is commonly performed by a single measurement of fluorescence but this fails to capture a profile of ROS generation over time. This study aimed to develop a real-time monitoring method to increase the utility of the assay, to incorporate cytotoxicity screening and to describe the combined effects of DCFDA and the ROS generator, Ter-butyl hydrogen peroxide (TBHP). Breast cancer MCF-7 cells were loaded with DCFDA (0-50 μM) for 45 min, and then exposed to TBHP (0-50 μM). Fluorescence was recorded according to three different schedules: every hour for 6 h, or once after 6 h or 24 h. Viability was assessed in a crystal violet assay and cell morphology was examined by microscopy. TBHP caused a time and dose-dependent increase in ROS and the magnitude of the fluorescent signal was affected by the loading concentration of DCFDA. Reading the fluorescence every hour for 6 h did not diminish the emission signal. The most sensitive and reliable combination for this ROS assay was 10 μM DCFDA with 25 μM TBHP; since higher concentrations of DCFDA compromised cell viability. In conclusion we adapted a single point ROS assay to enable production of a profile of ROS generation over an extended 6 h period, and related this to cell viability and morphology. en_US
dc.identifier.citation Figueroa, D., Asaduzzaman, M., & Young, F. (2018). Real time monitoring and quantification of reactive oxygen species in breast cancer cell line MCF-7 by 2ʹ,7ʹ– dichlorofluorescin diacetate (DCFDA) assay. Journal of Pharmacological and Toxicological Methods, 94, 26–33. https://doi.org/10.1016/j.vascn.2018.03.007 en
dc.identifier.doi https://doi.org/10.1016/j.vascn.2018.03.007 en
dc.identifier.issn 1056-8719
dc.identifier.uri http://hdl.handle.net/2328/38073
dc.language.iso en
dc.publisher Elsevier en
dc.rights Published by Elsevier Inc. en
dc.rights.holder Elsevier Inc. en
dc.rights.license CC-BY-NC-ND
dc.subject Reactive oxygen species en
dc.subject DCFDA en
dc.subject MCF-7 breast cancer cells en
dc.subject Cell viability en
dc.subject ROS assay optimisation en
dc.subject Real time monitoring en
dc.title Real time monitoring and quantification of reactive oxygen species in breast cancer cell line MCF-7 by 2′,7′–dichlorofluorescin diacetate (DCFDA) assay en
dc.type Article en
local.contributor.authorOrcidLookup Figueroa Gonzalez, Daniela: https://orcid.org/0000-0001-7317-7622
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