Lipid-ROS Assessment for Ferroptosis

The accumulation of lipid-ROS is a major feature of ferroptosis and can be used to infer the degree of ferroptosis [47 (link)]. Cells were treated with DMSO, anisomycin, or SB203580 plus anisomycin for 24 h or 12 h. Then, cells were incubated with 3 μM C11-BODIPY 581/591 (Invitrogen, USA) in serum-free DMEM medium for 30 min at 37°C in an incubator. Afterwards, cells were washed three times with serum-free medium, trypsinized, and resuspended in PBS before detected by flow cytometry. Lipid-ROS levels were evaluated by the fluorescence intensity of FITC channel, and the mean fluorescence intensities (MFI) of FITC were calculated for each sample. Experiments were performed in triplicate.