3.3.4. DCF-DA Assay Protocol

DCFDA is a fluorescent dye that measures the activity of hydroxyl radicals, peroxyl radicals, and other reactive oxygen species (ROS) within the cell. The DCFDA analysis protocol is based on the diffusion of DCFDA into the cell. It is then deacetylated from cellular esterases to a non-fluorescent compound, which is later oxidized by ROS to 2’,7’ -dichlorofluoroscein (DCF). The produced compound (DCF) is fluorescent and is detected by fluorescence spectroscopy at 485/535 nm (excitation/emission).

For the experimental procedure a standard solution of DCF-DA (20mM) in DMSO was prepared which was stored in the dark (−20 °C) until use. In a 96-well plate, cells were cultured to a density of 3 × 10⁴ cells/well.

The cells remained in the incubator (37 °C, 5% CO₂) for 24 h, after which an appropriate amount of H₂O₂ (2 mM) was added and the dish was returned for incubation for the next 2 h. This was followed by the addition of the test substances (100μM) and incubation for 24 h. At the end of the treatment time, all wells were washed with PBS, and DCF-DA solution (working concentration: 10 μM) (in 200 μL of nutrient) was added and incubated for 40 min.

After incubation, the medium with the DCF-DA was removed and replaced with FBS-free DMEM. The measurement of the absorption was made immediately after that in a plate reader at 495/530 nm (excitation/emission).

All samples were tested in triplicates, against a control (without effects), blank (without cells), and positive control (standard 100 μM Ascorbic acid solution was used).

The percentage of antioxidant capacity (%) was calculated according to the following Equation (6) evaluating the presence of ROS [42]: