4.13.2 Catalase (CAT) activity assay

Catalase (CAT) activity was evaluated by the catalase assay kit (MAK381, Sigma-Aldrich, Milan, Italy), according to the manufacturer´s instructions. RBCs were exposed to 50 mM AAPH for 1 h at 37°C with or without pre- or post-incubation with the anthocyanin extract (0.01 μg/mL) for 1 h at 37 °C. As the positive control, cells were exposed to 20 mM H₂O₂ for 30 min. After each treatment, the incubation medium was discarded and cells were washed in PBS 1X. Subsequently, cells were lysed in 0.2 mL catalase assay buffer and centrifuged at 10,000 for 15 min at 4 °C. The supernatant (50 µL) was incubated with 12 µL of 20 mM H₂O₂ for 30 min at 25 °C. Stop solution (10 µL) and developer reaction mix (50 µL) were added and the samples were incubated for 10 min at 25 °C. CAT activity was determined by reading the absorbance at 570 nm wavelength (Fluostar Omega, BMG Labtech, Ortenberg, Germany) after subtracting the background.