*4.6. Detection of Intracellular ROS*

The ROS-sensitive fluorescent dye, 2 ,7 -dichlorofluorescein diacetate (DCFDA; Sigma-Aldrich), was utilised to measure the intracellular ROS levels. SH-SY5Y cells (2.2 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/mL) were cultured in black 96-well plates in DMEM/F12 without phenol red. Then, 60–70% confluence cells were stimulated with H2O2 (300 μM) for 1 h in serum-free media and then washed twice with PBS, followed by a 30 min incubation with DCFDA (10 μM) in PBS. The cells were then rinsed with PBS twice. Finally, 200 μL PBS was added, and fluorescence was measured using 485 nm excitation and 535 nm in a fluorescence microplate reader (Molecular Device; Sunnyvale, CA, USA).

#### *4.7. Assessment of Caspase-3 Activity*

Caspase-3 activity was measured using a Colorimetric Caspase-3 Assay Kit (Sigma-Aldrich; St. Louis, MO, USA), as described previously [41]. The reaction mixture (total volume, 200 μL) was distributed in 96-well plates and incubated at 37 ◦C for 90 min. Absorbance values were measured at wavelengths of 405 nm in a Tecan Microplate Reader (Meilen; Zurich, Switzerland).
