Evaluating Antioxidant Efficacy in Cell Lines

To evaluate the efficiency of anti-oxidant activity, Caco-2, NCI-H441 and SHSY5Y cells were pretreated with BDVs (5 and 22 μg/mL) for 24 h. Then, in order to induce oxidative stress, Caco-2 and NCI-H441 cells were treated with 100 µM hydrogen peroxide (H₂O₂, Sigma-Aldrich, Cat# H1009) for 24 h^{27 (link),28 (link)}, while SHSY5Y cells were challenged with 100 µM 6-hydroxydopamine (6-OHDA, Sigma-Aldrich, Cat# H4381) for 24 h^{29 (link)}. Reactive oxygen species (ROS) production was evaluated by fluorimetry by using the 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA, Sigma-Aldrich, Cat# D6883)), as previously described^{28 (link)}. The dose and timing of H₂O₂ and 6-OHDA incubation with cells were chosen based on preliminary experiments that evaluated significant ROS production in the absence of overt reduced viability. Briefly, Caco-2, NCI-H441 and SHSY5Y cells were washed and then incubated with 10 µM H2DCFDA for 90 min at 37 °C and the fluorescent signal of DCF obtained from the conversion of H2DCFDA by intracellular ROS produced was measured at excitation/emission wavelengths of 485/530 nm using a Fluorescent Plate Reader (FilterMax F5 Multi-Mode Microplate Reader (Molecular Devices, Beckman Coulter, CA, USA). The results were expressed as mean fluorescence intensity (MFI). The assay was performed two times in quadruplicates.