Implementation of the FENIX assay to assess the anti-ferroptotic properties of test compounds

Ferroptosis is an iron-dependent form of regulated cell death characterized by the uncontrolled accumulation of lipid peroxides, primarily within phospholipid membranes. This oxidative process, driven by a radical-mediated chain reaction known as autoxidation, plays a central role in numerous pathological conditions including neurodegeneration, ischemia-reperfusion injury, and cancer. Pharmacological inhibition of lipid peroxidation by radical-trapping antioxidants (RTAs) has thus emerged as a promising strategy for therapeutic intervention. However, conventional 'antioxidant assays' such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) or oxygen radical absorbance capacity (ORAC) are inadequate for assessing reactivity toward lipid peroxyl radicals, limiting their utility in ferroptosis research. To address this gap, the Fluorescence-ENabled Inhibited autoXidation (FENIX) platform was developed as a physiologically relevant, high-throughput assay system for the mechanistic and kinetic characterization of lipid peroxidation inhibitors. FENIX-1 quantifies RTA activity by measuring rate constants for RTA-lipidperoxyl radical reactions initiated by a highly lipid-soluble radical initiator in liposomal systems wherein a fluorescent reporter is co-autoxidized along with the lipids. In FENIX-2 lipid peroxidation is initiated via iron-mediated decomposition of pre-formed phospholipid hydroperoxides, enabling the study of both RTA and non-RTA inhibitors, including iron chelators and peroxidase mimics. Together, these assays are complementary and mechanistically informative platforms for the identification and evaluation of inhibitors of lipid peroxidation and - by extension - ferroptosis. Here, we present a detailed overview and standardized protocols for both FENIX-1 and FENIX-2, with the aim of encouraging their application in lipid peroxidation research, ferroptosis modulation, and redox-targeted drug discovery.