Abstract
Lipid peroxidation of polyunsaturated fatty acid (PUFA) phospholipids induces necrotic cell death through compromised cell membrane integrity during ferroptosis. We established assays to investigate oxidoreductase-mediated oxidative rupture, specifically via NADPH-cytochrome P450 reductase (POR) and NADH-cytochrome b5 reductase (CYB5R1), of PUFA phospholipids in artificially generated protein-free liposomes. Liposome breakage was detected via Tb3+ liposome release and electron microscopy liposome morphology imaging. This protocol was also applied to other oxidoreductases with analogous functions and investigation of ferroptotic membrane damage in cell-free systems. For complete details on the use and execution of this protocol, please refer to Yan et al. (2020).