2.7. Determination of Radical Scavenging Activity at the Plasma Membrane Using CAA/DCFH-DA Bioassay

The CAA test is based on the use of a diacetate group (DA) which allows for the cell uptake of a DCFH-DA probe (2′, 7′-dichlorofluorescein-diacetate) through the plasma membrane. DCFH-DA is then cleaved by cellular esterases and DCFH is trapped within the cells. Cells are treated with a radical generator AAPH (2, 2′-azobis (2-amidinopropane) dihydro-chloride) to initiate oxidation. Peroxyl radicals produced at the plasma membrane convert non-fluorescent intracellular DCFH to its oxidized product DCF, which becomes fluorescent upon excitation [36]. The CAA test measures the decrease in cellular fluorescence intensity reflecting antioxidant effect or peroxyl radical scavenging activity in the plasma membrane [36]. Here, cells were incubated with samples (9 concentrations obtained by serial dilutions) for 1h at 37 °C in 5% CO2, in the presence of DCFH-DA (30 µM), followed by three washes, and treatment with AAPH (600 µM). Quercetin was used as a positive control. Fluorescence (RFU) was read every 5 min on a kinetic mode for as long as necessary. Dose–response curves were obtained according to the formula (control = cell culture medium):

Experiments were performed in triplicate, in culture medium without FBS, at least twice.