Lipid peroxidation detection

Lipid peroxidation was assessed based on the fluorescent lipid probe C11-BODIPY 581/591 (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4bora-3a,4a-diaza-s-indacene-3 undecanoic acid).¹⁹ Upon oxidation, the probe color changes from non-peroxidized (red fluorescence) to peroxidized (green fluorescence). Cells were seeded in a 96-well black/clear bottom plates (Greiner Bio One) and incubated for 24h at 37°C. Cells were then treated with the corresponding treatments (i.e. PunA, fer1, RSL3, Dara, or combinations thereof) for 4h before being incubated with 5 μM of C11-BODIPY 581/591 for 30 min at 37°C. Next, cells were washed with PBS and the red and green fluorescence levels were evaluated using a Spectra- max iD3 microplate reader (Molecular Devices) (red: λexcitation = 580 nm, λemission = 620 nm; green: λexcitation = 500 nm and λemission = 540 nm). The wavelengths were selected after optimization and spectral range detection. Data are expressed as relative fluorescence units determined by dividing the green fluorescence by the red fluorescence and calculating the ratio between the value obtained for the test condition and the one obtained for the control.