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.
Messner and Kowdley BMC Gastroenterology 2008 8:2 doi:10.1186/1471-230X-8-2