Characterization of non-transferrin-bound iron internalization in T51B liver epithelial cells. A. Non-heme iron content. Cells were left untreated (none) or treated with 200 μM ferric ammonium citrate (FAC) or with 200 μM FAC and 160 μM desferoxamine (FAC + dfo) for 5 days. Total non-heme iron (nmol/mg cell lysate protein) was determined by a ferrozine-based colorimetric assay as described under Methods. Values are reported as the means +/- s.e.m. of triplicate dishes (**p < 0.001 compared to control). B. Quenching of calcein fluorescence. Cells were pulsed with calcein-AM for 30 minutes, rinsed, and incubated for 2 days in complete cell media containing: (i) no addition, (ii) 200 μM FAC, or (iii) 200 μM FAC and 160 μM dfo. Identical fields for FITC fluorescence (left panels; corresponding to calcein signal) and phase contrast (right panels) are shown. C. Ferritin content. Cells were treated with 0, 200, or 500 μM FAC for 5 days and processed for western blots using antibodies specific for ferritin L, ferritin H, or GAPDH as gel loading control. Each experiment was performed at least twice with similar results.