2.4. Reactive Oxygen Species (ROS) Assay

The ROS Detection Assay Kit (BioVision, Milpitas, CA, USA) was used to measure the amount of intracellular ROS following the manufacturer’s protocol. Briefly, 10,000 HepG2 or A549 cells in 100 µL of culture medium/well were seeded on 96 flat-bottomed multi-wells and incubated overnight at 37 °C to allow adhesion. After 24 h, the medium was removed and cells were washed once with 100 μL ROS assay buffer, combined with 100 μL/well 1X ROS assay label, and incubated for 1 h at 37 °C. At the end, the ROS label solution was removed and cells were treated with the three highest concentrations of MZ (at 295.7, 29.6 and 2.96 μM), ZOX (at 463.4, 46.34 and 4.63 μM), vehicle as negative control (DMSO 0.8% and 0.2% for cells treated with MZ and ZOX, respectively), or H₂O₂ 100 μM as positive control in duplicated wells, and incubated for 24 h at 37 °C. Plates were read for green fluorescence from the bottom (485 nm excitation, 535 nm emission) by the Victor 3 Multilabel Reader (PerkinElmer). Three independent experiments were performed. After background subtraction, the fold-change of the fluorescence reading of treated cells with respect to vehicle control cells was calculated.