C11 bodipy 581 591 fluorescence probe

Cells were orderly stained with C11-BODIPY 581/591 fluorescence probe (1 μM, Invitrogen, C10445) and Hoechst 33342 (Invitrogen, H1399) for 30 min. The extent of lipid peroxidation was represented as the ratio of GFP(484/510 nm) to RFP (581/610 nm) fluorescence detected by the inverted fluorescence microscope (EVOS FL, AMG, USA) and quantified by ImageJ. Fluorescence was also measured by channel 488 Grn using the multiparameter flow cytometer (A60-Universal, Apogee, Britain), and the data were analyzed by FlowJo v10.8.1.
Accompanied by DFO chelating iron, Calcein-AM (1 μM, Invitrogen, C3099) was used to assess the labile iron pool (LIP). After incubation of Calcein-AM for 15 min, the fluorescence intensity at 496 nm (excitation wavelength) and 520 nm (emission wavelength) of initial and after iron chelation were measured by the microplate reader (Enspire, PE, USA) were recorded, respectively. The discrepancy between the latter and the former was indicated as the relative content of LIP.
The ratio of GSH/GSSG was tested by GSH and GSSG Assay Kit (Beyotime, S0053) according to the manufacturer’s instructions.

Cells were seeded at a density of 2.5 × 10⁵ per well on coverslips placed in a 6- well dish for overnight. After 24 h of Bap exposure, cells were washed twice in PBS and incubated in a serum-free medium containing 2 μM C11- BODIPY 581/591 fluorescence probe (Invitrogen) added to per well, and incubated at 37 °C with 5% CO₂ for 20 min. Coverslips were then washed twice with PBS and inverted onto microscope slides. Slides were imaged using a Nikon ECLIPSE 50i microscope.