*2.7. Measurement of Intracellular ROS Level*

Reactive oxygen species were detected in intact cells, according to Bergamini et al. [40]. Briefly, NLF and BR6 were seeded at the density of 1.5 <sup>×</sup> <sup>10</sup><sup>4</sup> cells per well in a 96-well plate and incubated for 24 h to allow adhesion. Then, cells were treated for 4 h with NS at concentrations ranging from 0.05 to 0.2 mg/mL or NF at 0.05 mg/mL or with the combinations of NS and NF. Cells were then incubated with 10 µM DCFDA (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h. To induce oxidative stress, cells were exposed to

150 µM tert-butyl hydroperoxide (TBH) in PBS for 30 min. Cells were then washed twice with PBS, and the fluorescence emission from each well was measured (λexc = 485 nm; λem = 535 nm) with a multi-plate reader (Enspire, Perkin Elmer, Monza, Italy). Data are reported as the mean ± SD of at least three independent experiments.
