Measurement of lipid peroxidation with BODIPY 581/591 C11 assay

The ability of SI to induce lipid peroxidation was investigated using lipid peroxidation sensor BODIPY 581/591 C11 (Thermo Fisher Scientific, Waltham, USA). BODIPY 581/591 C11 is a lipophilic fluorescent dye for indexing lipid peroxidation in cellular membranes^55,56. In brief, ARPE-19 cells were seeded in a 96-well plate at density of 8000 cells/well. After 24 h, cells were loaded with 2 μM BODIPY 581/591 C11 for 30 min at 37 °C followed by treatment with or without NaIO₃ for 0.5 h or 6 h. As a positive control, cells were treated with 100 μM tBH and 100 nM Heme for 0.5 h or 6 h. Then the rates of lipid peroxidation were measured using a Varioskan Flash multimode reader (Thermo Fisher Scientific, USA) with excitation/emission of 495/521 nm for the green signal (oxidized) and 575/600 nm for the red signal (non-oxidized). Oxidation of BODIPY 581/591 C11 is presented as a ratio between green fluorescence (oxidized) and total fluorescence (oxidized plus non-oxidized).