2.4. Reactive Oxygen Species (ROS) Production Assay

Early ROS generation was assessed using H₂-DCFDA, a fluorescent probe. The ROS generation assay was performed according to Zingales et al. [7]. Specifically, 30,000 cells/well were seeded in a 96-well plate with black walls. After 48 h, the medium was changed, and the cells were incubated for 20 min with 20 μM H₂-DCFDA (in cell culture medium). Then, the H₂-DCFDA solution was replaced with 200 μL/well of a medium containing ≤1% DMSO (control) or CIT at concentrations of 19.38, 25, and 38.75 μM. These concentrations were selected based on previous cytotoxicity assays. All tested concentrations were below the IC₅₀ values obtained. Fluorescence was measured at excitation and emission wavelengths of 485 and 535 nm, respectively, using a Wallace Victor2 1420 (PerkinElmer, Turku, Finland), with readings taken at intervals up to 120 min. The results are presented as the increase in fluorescence compared to the solvent control. The experiments were conducted independently twice, with 24 replicates each.

In addition, ROS production at 24 h of CIT exposure was also assessed. In this assay, after confluence, the SH-SY5Y cells were exposed to CIT by replacing the culture medium with fresh medium containing CIT at final concentrations of 25, 38.75, and 50 μM. After 24 h of exposure, the supernatant with CIT was removed, and each well received 200 μL of fresh medium with 20 μM H₂-DCFDA. The plates were incubated at 37 °C for 30 min, in darkness. Fluorescence measurements were determined at excitation and emission wavelengths of 485 and 535 nm, respectively, using a Wallace Victor2 1420 (PerkinElmer, Turku, Finland). The results were divided by the cell viability at each concentration and expressed in comparison to the control. The experiments were conducted independently in duplicate, with 24 replicates each.