Comparative effects of chelators on cytosolic LIP as analyzed in various cell types probed with the ROS-sensitive oxidizable probe CDDHCF-DA. HepG2, J774, and H9C2 cells grown to confluence in 96-well culture plates were preincubated for 18 hours with the indicated chelator (0 = control, 20 or 100 μM in growth medium). Prior to fluorescence analysis, the cells were loaded with the nonfluorescent oxidizable CDDHCF-DA (10 μM), which generates the free acid intracellularly. After cells were washed and resuspended in buffered saline containing 10 mM glucose and 0.5 mM probenecid, fluorescence was followed in a fluorescence plate reader (excitation, 485 nm; emission, 515 nm) at 37°C: H₂O₂ (20 μM) (first) and chelator (second) were added sequentially (5-6 minutes apart). The slopes of fluorescence rise with time were calculated (as shown in Figure 2, inset) and are given relative (normalized) to the value of the control (no chelator added) ± SEM. “Online” comprises systems that were preincubated with no chelator, while “after 18 hours” comprises systems that were preincubated for 18 hours with the indicated chelator (0, 20, or 100 μM). The slopes of the respective controls (prior to normalization) were as follows for H9C2, HepG2, and J774 cells (in arbitrary fluorescence units/min): 980 ± 30, 425 ± 45, and 405 ± 33, respectively. The horizontal broken lines represent, for each cell type, the basal oxidation level that is not contributed by metal, revealed by addition of 200 μM ICL670.