Transfer of iron from extracellular DFP-iron complexes to mitochondria. H9C2 cells loaded with the mitochondrial iron-sensor RPA were incubated with or without DFP-Fe (15:5 μM) and epifluorescence microscopic images were recorded every 5 minutes under settings for rhodamine. Representative fields of initial cell fluorescence at time 0 (A,C) and after incubation for 1 hour in the presence (B) or absence (D) of 5 μM DFP-Fe complex. Magnification was ×600; oil objective was a (Plan Apo) 60×/1.40 NA. (E) Mean fluorescence values in relative units (r.u.) plus or minus SD of 5 cells per field calculated for each time-point image and normalized to the initial fluorescence, representing cells incubated without (None) and with 5 μM Fe-complex (DFP-Fe or NTA-Fe); the lines denoted as ΔDFP-Fe and ΔNTA-Fe were corrected for spontaneous decay given by the control (None). Arrow indicates time the Fe complex was added. (F) Effect of various iron chelates on the fluorescence (f normalized to initial value, f₀, in r.u.) of 0.5 μM RPA in solution (HBS buffer). The iron chelates all contained 5μM Fe complexed to NTA (1:3 ratio) in the absence (■) and presence (□) of 100 μM ascorbate (Fe:NTA, Fe:NTA + ASC) or to DFP, with 100 μM ascorbate, at DFP:Fe ratios of 3:1 (●), 5:1 (○), and 7:1 (▵). The values of fluorescence intensity are means of triplicate samples run in parallel, plus or minus SEM.