Chelator access to endosomal labile iron in H9C2 cells and cardiomyocytes, as revealed by endocytosed CALG-Fe complexes. H9C2 or cardiomyocytes were incubated with fluorescence-quenched CALG/Fe (1:1 ratio, 30 μM) for 1 hour at 37°C in HEPES-buffered DMEM medium, then washed and incubated at 37°C in the same medium. Fluorescence (485 nm excitation, 520 nm emission) was monitored by fluorescent microscopy (×60 objective). The images shown are of H9C2 cells, selected from a time sequence of 15 images recorded at 2-minute intervals: A was taken at 2 minutes (prior to chelator addition) and B at 28 minutes (18 minutes after addition of 50 μM DFR). C is a merged picture of fluorescence and phase contrast of image B. D represents mean fluorescence values of 4 cells/field calculated for each time-point image and normalized to the basal fluorescence prior to addition of chelator, in relative units (ru). The chelators DFO (triangles), DFP (circles), and DFR (squares) were added at 50 μM concentrations at 10 minutes, as indicated by arrow. H9C2 cells are indicated by closed symbols; primary cardiomyocytes, by open symbols.