3.8. Cellular Antioxidant Activity (CAA) Assay

Cellular antioxidant measurements were carried out following the procedure employed by Kellett et al. [50]. Advanced DMEM medium supplemented with 10% endotoxin-free, heat-inactivated fetal bovine serum (FBS), 1% L-glutamine and 1% penicillin-streptomycin were used as the culture media. The quercetin standard was prepared in the concentration range from 25 to 200 μM. Both MCE and ACE were dissolved in 95% (v/v) ethanol and tested at concentrations of 10 and 25 mg of blackberry f.w. eq./mL. The cells were cultured in a Corning Costar^® 96-well plate and incubated at 37 °C with 5% (v/v) CO₂ until confluence. On the day of analysis, the media was removed and the cells were washed three times with PBS. The working DCFH-DA solution (50 μL) was added, followed by 50 μL of the culture media containing the quercetin standard, or a blackberry phenolic extract, or blanks (i.e., culture media containing the equivalent amount of ethanol). The cells were incubated at 37 °C for 60 min. After incubation, the media was removed, and cells were washed again with PBS (3×). One hundred microliters of 600 μM ABAP prepared in HBSS were added to each treatment well as a free-radical generator, and the plate was immediately transferred to the BMG FLUOstar Omega microplate reader (BMG LABTECH Inc., Cary, NC, USA) for measurement. The excitation/emission wavelength was set at 485/538 nm for fluorescence signal detection. A total of 13 cycles of fluorescent response was collected and the area under the curve (AUC) was calculated using MARS Data Analysis Software (BMG LABTECH). A reduction in fluorescence was calculated using the equation:

where, A_s and A_b referred to AUC of samples and blank, respectively.