Measurement of ROS production

The effect of allicin on ROS production in HUVECs was evaluated by fluorometric assay using DHE as a probe for the presence of superoxide [14]. After preincubation for 2 h with allicin at the indicated concentrations, HUVECs were incubated with DHE for 1 h, followed by incubation with ox-LDL for 2 h. The fluorescence intensity was measured at 540-nm excitation and 590-nm emission (before and after exposure to oxLDL) using a fluorescence microplate reader (Labsystems). The percentage increase in fluorescence per well was calculated by the formula [(Ft₂ − Ft₀)/Ft₀] × 100, where Ft₂ is the fluorescence at 2 h of ox-LDL exposure, and Ft₀ is the fluorescence at 0 min of oxLDL exposure.