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

Thumbnail Image
Figueroa Gonzalez, Daniela
Asaduzzaman, Mohammad
Young, Fiona Margaret
Journal Title
Journal ISSN
Volume Title
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.
Published by Elsevier Inc. This manuscript version is made available under the CC-BY-NC-ND 4.0 license: 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
Reactive oxygen species, DCFDA, MCF-7 breast cancer cells, Cell viability, ROS assay optimisation, Real time monitoring
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.