Effect of chelators on catalytically active iron in the cytosol and mitochondria of H9C2 cardiomyocytes. (A-B) Detection in mitochondria: The cells were loaded with DHR 50 μM (in HBS + 10 mM glucose) for 10 minutes at 37°C and washed, followed by epifluorescence (excitation, 488 nm; emission, 520 nm) at 1- to 2-minute intervals (A). After a baseline was established, agents were added at the indicated times: 35 μM H₂O₂, 100 μM chelator (ICL670 or DFP), or 50 μM BAPTA-AM. “None” indicates cells that received no treatment other than DHR, and “no chelator” refers to cells treated with H₂O₂ but with no chelator. (B) The image represents DHR-loaded cells treated with H₂O₂ but without chelator (fluorescence and phase merged). (C-D) Detection of oxidized CDCF in cytosol: The cells were preincubated with FeSO₄ for 1 hour at 37°C in HBS + 10 mM glucose, washed, resuspended in the same medium with no iron source but containing 20 μM CDDHCF-DA (the reduced precursor of CDCF), and examined by epifluorescence (excitation, 488 nm; emission, 520 nm) at 1- to 2-minute intervals (bottom left; fluorescence intensity given as relative units r.u.). Labels are as indicated for mitochondria, except that the images refer to CDDHCF-DA of cells prompted with Fe but treated with no chelator (fluorescence and phase merged) (D).