Cells were seeded at 3 × 105 cells per well in 6-well plates. Next day, cells were treated with Erastin (10 μM) and/or hemin (5 μM), CORM (10 μM), biliverdin (10 μM) for 8 hours. After 8 hours, cells were incubated with 2 μM CellROX® Deep Red (cytosolic ROS) or 2 μM C11-BODIPY581/591 (lipid peroxidation) (Invitrogen, Life Technologies, Grand Island, NY) for 30 minutes at 37°C in the dark. After 30 minutes of loading, unincorporated dye was removed by washings with 2% FBS containing PBS. Samples were then centrifuged at 1000 rpm for 3 minutes and the pellets were resuspended in 500 μL of 2% FBS containing PBS. Measurements were performed on a FACSCalibur (Becton Dickinson, San Jose, CA) flow cytometer.
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