Reactive Oxygen Species Determination Using DCF-DA Method

Intracellular formation of ROS was assessed using oxidation sensitive dye 2',7'-dichlorofluorescein diacetate (DCF-DA; Sigma-Aldrich, Darmstadt, Germany) as a substrate. DCFH-DA is a non-fluorescent compound that is passively diffused into cells where is hydrolyzed by esterases to yield non-permeable DCFH. In the presence of ROS, DCFH is oxidized to the fluorescent DCF. MCF-7 cells or RAW 264.7 macrophages were seeded into 96-well microtiter plates at a density of 25 × 10³/well and pre-incubated for 24 h at 37°C. The cells were then stimulated with the different concentrations of AgNPs for 24 h. Subsequently, the cells were washed twice with phosphate-buffered saline (PBS) and incubated in the darkness with 20 μM DCFH-DA (200 μl per well) for 45min at 37°C. The fluorescence was measured using a Synergy HT Multi-Mode Microplate Reader (BioTek; Winooski, VT, United States) with excitation at 485 nm and emission at 528 nm. Culture media backgrounds containing AgNPs concentration were subtracted from the corresponding wells with cells. The ROS level was calculated according to a formula, as given above for the MTT assay, in which the absorbance values were replaced with fluorescence values. All data were from three independent experiments with five wells for each experiment. The results were expressed as the fold change relative to equivalent control untreated cells.