2.7. Amplex Red hydrogen peroxide assay

Extracellular H₂O₂ levels were assessed using the Amplex Red Hydrogen Peroxide Assay Kit, according to manufacturer's instructions (Invitrogen, #A22188). Cell culture medium was collected 24 and 48 h after indicated treatments and incubated with 100 µM Amplex Red reagent containing 0.2 U/mL horseradish peroxidase for 30 min at RT. Fluorescence was measured using a microplate reader (ex: 530 nm, em: 590 nm), and concentrations calculated based on standard curves.