3.5.2. Antioxidant Cell Assay Using 2′,7′-Dichlorofluorescin-Diacetate (DCFH-DA)

Antioxidant activity was evaluated using the DCFH-DA assay as described by Girard-Lalancette et al. [37], with some modifications. Briefly, Human Skin WS-1 fibroblasts (ATCC CRL-1502, ATCC, Manassass, VA, USA) were plated in 96 microwell plates at 10,000 cells per well and incubated for 24 h at 37 °C and 5% CO₂. The cells were washed with 150 μL Hank’s balanced salt solution (HBSS) at pH 7.4 and incubated for 30 min with 100 μL HBSS (pH 7.4) containing 5 μM DCFH-DA (Sigma–Aldrich, Oakville, ON, Canada). The cells were then washed again with 150 μL HBSS. To assess antioxidant activity, the cells were incubated either with a growing concentration of enriched fraction from A. nudicaulis, trolox or quercetin, in the absence or presence of 200 μM tert-butylhydroperoxide (tBH). Fluorescence was measured after 1 h and 4 h on the Fluoroskan Ascent FL™ automated plate reader using an excitation wavelength of 485 nm and an emission wavelength of 530 nm.