Chelator access to nuclear and cytosolic labile cell iron pools as sensed by histone-conjugated CALG and rhodamine B (CALG-H-R). H9C2 (A) and primary cardiomyocytes (B) were initially loaded for 1 hour with CALG-H-R (12 μM) at room temperature and washed as described in “Materials and methods.” The fluorescence microscopy images show the predominantly nuclear and partially cytosolic distribution of the probe, which was found to be maintained for over 24 hours. The cells loaded with CALG-H-R were incubated for 24 hours in the presence of the indicated additives. (C) CALG-H-R loaded H9C2 cells in complete growth medium supplemented with FAC (50 μM), DFR (50 μM), DFO (100 μM)), DFP (50 μM), or with no addition (con). (D) CALG-H-R loaded primary cardiomyocytes incubated in 30% human serum containing 2 μM LPI final concentration (LPI), which was supplemented with 10 μM DFO (LPI-DFO) or 50 μM DFR (LPI-DFR) or 50 μM DFP (LPI-DFP). Panels C and D depict the LCI values for nuclear (N, ▪) and cytosolic (C, ▨) compartments estimated by analysis of images obtained before and after addition of excess permeant chelator (DFR 200 μM, applied for the purpose of attaining maximal recovery of fluorescence) and normalized to the initial fluorescence. The insets show the kinetics of the changes in nuclear fluorescence in response to 200 μM DFR (indicated by arrow), based on a mean of 3 to 5 cells in a field. The various preincubation conditions are indicated for each trace. Error bars indicate SD (n = 4).