S0103

ELISA detection kits for total superoxide dismutase (SOD, S0103, Beyotime Institute of Biotechnology, Nantong, China), total glutathione peroxidase (GPx, S0058, Beyotime Institute of Biotechnology), and malondialdehyde (MDA, S0131S, Beyotime Institute of Biotechnology) were used for measurements. Assays were performed following the manufacturer’s instructions. The brain tissues were isolated on day 7 and placed in 0.9% cold saline. Samples were homogenized and centrifuged at 3000 r/minute for 15 minutes. The supernatant was collected for ELISA, and samples were analyzed using a Synergy™ HT multimode microplate reader (Biotek, Shanghai, China).
To measure MDA, SOD and GPx activities in SH-SY5Y cells, cells were seeded at 1 × 10⁵ cells/well in 96-well plates for 24 hours, followed by co-treatment with 100 μM hemin at various concentrations (1, 10 and 100 nM) of WFA or ferrostatin-1 (fer-1, 10 μM) for 24 hours. The cells were collected using a rubber scraper and centrifuged at 1000 × g for 10 minutes at 4°C. Cell pellets were lysed in 500 μL of 5% (w/v) metaphosphoric acid. Cellular lysate was centrifuged at 13,000 × g for 5 minutes at 4°C. Supernatants were collected for measurement of GSH-Px, SOD and MDA activities using a Synergy™ HT multimode microplate reader (Biotek).