Fe(II) Supplementation and Chelation Experiments

250 μM DFO or 1 mM BPS was added to DMEM media containing 10% FBS and glutamax in chambers containing cells and incubated at 37 °C for 8 h. At this point, media in these wells was replaced with 250 μM DFO or 1 mM BPS containing DMEM media (without FBS and glutamax) and incubated for 90 min at 37 °C. DMEM media in nontreated wells was aspirated from chambers containing cells and this was replaced with DMEM media containing 100 μM FAS (prepared from a 20 mM FAS solution in water) or DMEM media alone and this was incubated for 90 min at 37 °C. After 90 min, DMEM media was aspirated and cells were washed one time with 500 μL HBSS. Then 500 μL HBSS containing 10 μM FIP-1 (diluted from 5 mM stock) was added to each well and this was incubated at 37 °C for 90 min. At this point, buffer was removed and each well was washed 2× with 500 μL HBSS. Then 500 μL of HBSS were added and snapshot images were taken. Cells were then incubated with 1 μM Hoechst 33342 at 37 °C for 10 min prior to imaging nuclear staining.