Comparing qualitative and quantitative spectroscopic techniques for the detection of the effect of direct iron loading of mammalian cell cultures.

Abstract

Iron overload augments diseases of the liver and microorganism infection as well as deregulates the immune system. In vitro analysis of the effects of iron loading and its chelation involves determining the amount of iron constituting overload, which metal sources and cell lines to use and reliable assay methods. The uptake of 500 microM FeSO4 or FeEDTA by CEMss, U937 or leukocytes was confirmed by inductively coupled plasma-atomic emission spectroscopy (ICP-AES). Excess iron increased CEMss viability (assessed by MTT, XTT, Trypan- and Alamar Blue) by an average of 18% (P = 0.034). Flow cytometry indicated dye-viable cells to be undergoing apotosis/necrosis while still confirming an increase (9%, P < 0.001) in excess iron-induced viability. The iron chelator desferioxamine (DFO) when added in addition to Fe, reversed the effects of excess iron (and vice versa) and had detrimental effects when used on its own (33% inhibition of viability as measured by dyes and 10.85%; P = 0.0427 assessed by flow cytometry). The 4 dyes demonstrated different levels of sensitivity in detecting the influence of iron or DFO but gave a related, qualitative picture while flow cytometry and ICP-AES data was more quantitative.