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

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, Terbutyl hydrogen peroxide (TBHP). Breast cancer MCF-7 cells were loaded with DCFDA (0-50 mu M) for 45 min, and then exposed to TBHP (0-50 mu M). 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 mu M DCFDA with 25 mu M 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.