Figure 1.
Figure 1. Effect of chelators on cytosolic LIP: time dependence of intracellular chelation of resident labile iron as analyzed in cells probed with the iron-sensitive CALB. HepG2 cells were preincubated for 0.5 hours (short term) or 12 hours (long term) with the indicated chelator (50 μM in growth medium), and prior to epifluorescence analysis (excitation, 385 nm; emission, 430 nm) they were loaded with CAL-B via its AM precursor. After washing with buffered saline, the cells were followed by epifluorescence microscopy under perfusion in DMEM-Hepes-buffered medium (with no phenol red) at 37°C. At the indicated times, the cells were supplemented with 50 μM SIH in order to obtain maximal recoverable fluorescence. (A) Snapshots of images of control cells preincubated 12 hours with no chelator (basal) and following a 10-minute treatment with SIH (in order to attain maximal recoverable fluorescence). (B) LIP analysis of cells treated for 12 hours with no chelator (control) or with the indicated chelator (50 μM). The fluorescence intensity values are mean values obtained at each time point from 5 different cells (in arbitrary units = a.u.); those corresponding to treatment prior to the addition of SIH (50 μM) denote the basal level of fluorescence. The solid vertical arrow represents the total fluorescence recovered in control cells by addition of SIH. The mean fluorescence intensity values are normalized to those associated with basal levels of control, and the difference between them and those attained by addition of SIH provides a measure for the cell chelatable iron, which we refer to as the labile iron pool (= LIP).20 • indicates control; ○, DFP; □, DFO; and *, ICL. The approach was applied to cells treated with chelators for either 12 hours (D) or 0.5 hours (C), with the slashed areas depicting the basal levels after 12-hour or 0.5-hour incubation and the black areas those attained with SIH (representing the residual LIP for each treatment). The bars depict standard errors of mean fluorescence intensity values obtained from 5 cells per field.

Effect of chelators on cytosolic LIP: time dependence of intracellular chelation of resident labile iron as analyzed in cells probed with the iron-sensitive CALB. HepG2 cells were preincubated for 0.5 hours (short term) or 12 hours (long term) with the indicated chelator (50 μM in growth medium), and prior to epifluorescence analysis (excitation, 385 nm; emission, 430 nm) they were loaded with CAL-B via its AM precursor. After washing with buffered saline, the cells were followed by epifluorescence microscopy under perfusion in DMEM-Hepes-buffered medium (with no phenol red) at 37°C. At the indicated times, the cells were supplemented with 50 μM SIH in order to obtain maximal recoverable fluorescence. (A) Snapshots of images of control cells preincubated 12 hours with no chelator (basal) and following a 10-minute treatment with SIH (in order to attain maximal recoverable fluorescence). (B) LIP analysis of cells treated for 12 hours with no chelator (control) or with the indicated chelator (50 μM). The fluorescence intensity values are mean values obtained at each time point from 5 different cells (in arbitrary units = a.u.); those corresponding to treatment prior to the addition of SIH (50 μM) denote the basal level of fluorescence. The solid vertical arrow represents the total fluorescence recovered in control cells by addition of SIH. The mean fluorescence intensity values are normalized to those associated with basal levels of control, and the difference between them and those attained by addition of SIH provides a measure for the cell chelatable iron, which we refer to as the labile iron pool (= LIP).20  • indicates control; ○, DFP; □, DFO; and *, ICL. The approach was applied to cells treated with chelators for either 12 hours (D) or 0.5 hours (C), with the slashed areas depicting the basal levels after 12-hour or 0.5-hour incubation and the black areas those attained with SIH (representing the residual LIP for each treatment). The bars depict standard errors of mean fluorescence intensity values obtained from 5 cells per field.

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