Figure 3.
Figure 3. Effect of iron-loaded sera and chelators on cytosolic labile cell iron pools of primary cardiomyocytes. Cardiomyocytes grown for 6 days in culture were incubated for 20 hours in media containing either 30% normal human serum or 30% serum from an iron-overloaded patient containing (final) LPI of approximately 2 μM or in medium containing 10% fetal calf and 10% horse serum and 50 μM FAC. Cells were loaded with CALB-AM in DMEM medium without serum or pH indicator dye as described in “Materials and methods,” and LCI was either estimated by epifluorescence microscopy and digital image analysis (A, B) or quantified in a fluorescence plate reader (C). The upper panel depicts CALB fluorescence in cells pre-incubated for 20 hours with 50 μM FAC in growth medium, then loaded with CALB and photographed before (A) and after (B) addition of 50 μM SIH. The 45% rise in fluorescence elicited by SIH corresponds to a LCI pool of 2.5 ± 0.8 μM, determined in a fluorescence plate reader, as described previously.41 The lower panel (C) depicts the values of LCI pools based on measurements in a fluorescence plate reader. The bars represent LCI pools in cells incubated for 20 hours in growth medium containing FAC: 50 μM in standard growth medium with 10% fetal calf serum, 10% horse serum; LPI: 30% LPI-containing serum from an iron-overloaded patient with no additions; DFO-pre: 30% LPI-containing serum supplemented with 10 μM DFO from the onset of 20 hours of incubation; DFO-post, DFP-post, and DFR-post: 30% LPI-containing serum, which was supplemented with 10 μM DFO or 100 μM DFP or 100 μM DFR at the conclusion of the 20-hour preincubation, 20 minutes before loading with CALB-AM. LCI denotes mean labile cell iron pool values with standard deviations obtained with 5 experimental cell systems run in parallel, calculated as mean intensity values immediately before and 10 minutes after addition of 50 μM SIH, as described elsewhere.41 The dotted line represents the LCI of cells exposed to standard growth medium containing 10% fetal calf serum and 10% horse serum.

Effect of iron-loaded sera and chelators on cytosolic labile cell iron pools of primary cardiomyocytes. Cardiomyocytes grown for 6 days in culture were incubated for 20 hours in media containing either 30% normal human serum or 30% serum from an iron-overloaded patient containing (final) LPI of approximately 2 μM or in medium containing 10% fetal calf and 10% horse serum and 50 μM FAC. Cells were loaded with CALB-AM in DMEM medium without serum or pH indicator dye as described in “Materials and methods,” and LCI was either estimated by epifluorescence microscopy and digital image analysis (A, B) or quantified in a fluorescence plate reader (C). The upper panel depicts CALB fluorescence in cells pre-incubated for 20 hours with 50 μM FAC in growth medium, then loaded with CALB and photographed before (A) and after (B) addition of 50 μM SIH. The 45% rise in fluorescence elicited by SIH corresponds to a LCI pool of 2.5 ± 0.8 μM, determined in a fluorescence plate reader, as described previously.41  The lower panel (C) depicts the values of LCI pools based on measurements in a fluorescence plate reader. The bars represent LCI pools in cells incubated for 20 hours in growth medium containing FAC: 50 μM in standard growth medium with 10% fetal calf serum, 10% horse serum; LPI: 30% LPI-containing serum from an iron-overloaded patient with no additions; DFO-pre: 30% LPI-containing serum supplemented with 10 μM DFO from the onset of 20 hours of incubation; DFO-post, DFP-post, and DFR-post: 30% LPI-containing serum, which was supplemented with 10 μM DFO or 100 μM DFP or 100 μM DFR at the conclusion of the 20-hour preincubation, 20 minutes before loading with CALB-AM. LCI denotes mean labile cell iron pool values with standard deviations obtained with 5 experimental cell systems run in parallel, calculated as mean intensity values immediately before and 10 minutes after addition of 50 μM SIH, as described elsewhere.41  The dotted line represents the LCI of cells exposed to standard growth medium containing 10% fetal calf serum and 10% horse serum.

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