MDA-MB-468 cells were seeded at low (2000 cells/cm2) or high (10,000 cells/cm2) cell density in DMEM-F12 in the presence or absence of 50 nM sodium selenite. After a 48-hour incubation, cells were incubated with 1 μM BODIPY 581/591 C11 (Thermo Fisher Scientific) for 15 min. Cells were washed three times with phosphate-buffered saline (PBS), trypsinized, and resuspended in PBS with EDTA (ethylenediaminetetraacetic acid) and DAPI (4′,6-diamidino-2-phenylindole) before fluorescence-activated cell sorting (FACS) analysis (excitation, 488 nm). Lipid peroxidation was assayed in 10,000 events by the change in fluorescence and represented as the ratio Em510nm/Em590nm. As a positive control for lipid peroxidation, cells seeded at high cell density were exposed to 50 μM t-butOOH for 1 hour and analyzed by FACS, as described, or incubated for an additional 23 hours to assay the effect on cell survival.

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