Lipid peroxidation generation detection

PC12 cells (1×10⁵/well) were seeded in 6-well plates overnight, and the cells were treated as follows: PC12 cells were transfected with miR-335 mimic, miR-335 inhibitor, miR-335 NC, or siRNA-FTH1, respectively, and then stimulated with 6-OHDA (50 µM) for 24 h; PC12 cells were treated with Erastin (cat. no. HY-15763, MedChem Express), Fer-1 (cat. no. HY-100579, MedChem Express), DFO (cat. no. HY-B0988, MedChem Express) or Z-VAD-FMK (cat. no. HY-16658B MedChem Express) for 24 h, respectively, and then stimulated with 6-OHDA (50 µM) for 24 h. The cells were incubated with 10 µM C11-BODIPY (581/591; cat. no. D3861, Thermo Fisher Scientific, Inc.) for 30 min at 37°C, then washed with PBS 3 times. Cells were harvested with trypsin (cat. no. 15050-065, Gibco; Thermo Fisher Scientific, Inc.), suspended in fresh medium, and analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Inc.). Fluorescence was measured by acquiring green (484/510 nm). The butadienyl portion of C11-BODIPY581/591 was oxidized, resulting in the 510 nm fluorescence emission peak. The data were analyzed using FlowJo v10 software.