C11 bodipy 581 591 lipid peroxidation sensor dye

For all imaging performed in this paper, cells were seeded in an 8-well chamber (Nunc, Roskilde, Sjælland, Denmark) at a density of 3 × 10⁴ cells/well in 1% BSA RPMI. If cells were adherent they were left overnight to adhere to wells. All imaging of live cells was performed at the La Trobe Bioimaging Platform on a Zeiss 800 Confocal Laser Scanning Microscope (Zeiss, Oberkochen, Germany) using 63× oil immersion at 37 °C and 5% CO₂. The duration of imaging is noted in figure legends for respective experiments. Some assays involved the use of fluorescent dyes, including To-Pro-3-APC (Life Technologies), C11-BODIPY 581/591 lipid peroxidation sensor dye (2.5 μM) (Invitrogen, Waltham, MA, USA) and 4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene (BODIPY 493/503) (0.1 mg/mL) (Thermo Fisher Scientific). Images were processed by using software by Zen Image Analysis (Zen Blue Edition version 10.1.19043, Jena, Germany).

To investigate the ability of GS-9 to induce lipid peroxidation, the C11-BODIPY 581/591 lipid peroxidation sensor dye (Invitrogen, Waltham, MA, USA) was used. This dye is sensitive to oxidation such that upon peroxidation by ROS in cells it causes a shift in its fluorescence emission from red (∼590 nm) to green (∼510 nm). Jurkat and A549 cells were seeded at a density of 1.5 × 10⁴ cells/well and 2 × 10⁴ cells/well, respectively, in a 96-well plate in culture media. A549 cells were left to adhere overnight (37 °C and 5% CO₂). Cells were then treated with GS-9 at concentrations listed in the figures with or without the presence of Ferrostatin-1 and incubated overnight. Before analysis, A549 cells were trypsinized and lifted before being stained with 2.5 μM C11-BODIPY 581/591 for half an hour. As described above, samples were analysed by flow cytometry.