Effect of chelators on cytosolic LIP: time dependence of intracellular chelation of resident labile iron as analyzed in cells probed with the ROS-sensitive probe CDDHCF-DA. H9C2 cells were preincubated for either 0.5 or 12 hours with the indicated chelator (0 = control or 50 μM in growth medium), and prior to fluorescence analysis were loaded with the nonfluorescent oxidizable DCDHF (20 μM). After washing and resuspension in buffered saline supplemented with the same concentration of chelator, the systems were followed by epifluorescence microscopy (excitation, 485 nm; emission, 515 nm) under perfusion at 37°C, and H₂O₂ (35 μM) was added at the indicated time, except in the cases marked “none.” The pictures (top) represent snapshots taken from control cells incubated with no chelator for 12 hours, and the inset represents the fluorescence tracings obtained with cells incubated with chelator for 12 hours (F/F0 is the fluorescence intensity of each experimental point normalized to the value at time zero). The bar graph provides the rates of fluorescence changes with time (= slopes calculated from the time courses shown in the inset given as relative fluorescence units F/F0 per second) for both 0.5- and 12-hour treatment with chelator.