Effect of ELT and iron chelators on intracellular ROS generation and cell function (insulin production) in iron-loaded cells. The time-course for ROS inhibition by ELT and other chelators are shown in (A) HuH7 or (B) H9C2 cells. Cells were iron-loaded and then rinsed 4 times, as described earlier. Chelators were then added, and the rate of change of ROS production was recorded as fluorescence change (excitation at 504 nm, emission at 526 nm) continuously over the course of 1 hour in the plate reader at 37°C. DFO, DFP, and DFX were used at 10 μM IBE, and ELT at 10 μΜ. The rate of ROS production was compared between chelator-treated and chelator-untreated cells. Data shown are readings from individual plates. ROS rate inhibition at 1 hour with CP40, DFO, DFP, DFX, and ELT is shown in (C) HuH7 and (D) Η9C2 cells at 10 µM IBE for each chelator and 10 µM ELT and 33 µM IBE CP40. In both cell types, 10 µM ELT shows greater inhibition of ROS than other chelators at the same concentration in both HuH7 and H9C2 cells. The extracellular hydroxypyridinone chelator, CP40, had no effect on ROS. (E) Effect of chelator treatment during a 90-minute period in RINm5F cells on ROS generation is shown. Results are the mean ± SEM of 4 observations in 1 experiment. (F) Effect of iron loading in RINm5F cells with 2 changes of RPMI media containing 10% to 25% FBS on insulin secretion. After treatment, cells were challenged with Kreb’s Ringer buffer containing glucose and insulin concentration in the supernatant determined as described in “Materials and methods.” (G) Effect of chelation treatment on insulin production in RINm5F cells iron-loaded with two 10-hour changes of RPMI media containing 25% FBS. *P < .05; **P < .01 compared with control. Results are the mean ± SEM of 3 observations in 1 experiment.