Colorimetric ferrozine-based assay for the quantitation of iron in cultured cells

doi:10.1016/j.ab.2004.03.049

Analytical Biochemistry

Volume 331, Issue 2, 15 August 2004, Pages 370-375

https://doi.org/10.1016/j.ab.2004.03.049 Get rights and content

Abstract

The ferrozine-based colorimetric assay described here permits the quantitation of iron in cultured cells in amounts ranging between 0.2 and 30 nmol. Ferrous and ferric iron were detected equally well by the assay and the accuracy was unaffected by other divalent metal cations. This colorimetric assay was used to study iron accumulation in brain astrocytes that had been cultured in 24-well dishes. Iron complexed to cellular proteins was made accessible to ferrozine by treatment of cell lysates with acidic KMnO₄ solution. The basal amounts of iron in untreated astrocyte cultures were approximately 10 nmol iron per mg protein. Incubation of the cells with ferric ammonium citrate caused the total cellular iron content to increase in a concentration-dependent manner. The estimates of cellular iron content that were obtained with the ferrozine-based assay did not differ from those determined by atomic absorption spectroscopy. The colorimetric assay described here provides a sensitive, cheap, and reliable method for the quantitation of intracellular iron and for the investigation of iron accumulation in cultured cells.

Section snippets

Chemicals

The assay described here was developed, refined, and extensively characterized in Melbourne (Australia). These data were confirmed and extended with AAS in Tübingen (Germany). Therefore, the Australian and German sources of the chemicals are listed here. Dulbecco's modified Eagle's medium (DMEM) was obtained from Gibco (Invitrogen, Melbourne) and from Life Technologies (Eggenstein, Germany). Fetal calf serum (FCS) was from CSL (Melbourne) and from Serva (Heidelberg, Germany). Streptomycin

Results and discussion

A ferrozine-based colorimetric assay was tested for its potential to quantify the total iron content of cultured astrocytes. Ferrozine forms a complex with ferrous iron that strongly absorbs light at 550 nm [8]. After application of the ferrozine and ascorbate-containing reaction mixture to FeCl₃ solutions, the absorbance at 550 nm rapidly increased to maximal values within 10 min and then remained stable for at least 60 min (data not shown). In the range between 0.2 and 30 nmol iron the absorbance

Acknowledgements

Much of the research in this paper was supported by a Senior Research Fellowship awarded to R.D. by NeuroSciences Victoria. We thank the Department of Psychology, Monash University for financial support, Professor U. Weser and Dr. H.J. Hartmann (Tübingen) for giving us the opportunity to use their atomic absorption spectrometer, and Dr. G. Bishop (Monash University) for helpful comments on the manuscript.

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Colorimetric ferrozine-based assay for the quantitation of iron in cultured cells

Abstract

Section snippets

Chemicals

Results and discussion

Acknowledgements

J. Inorg. Biochem.

Methods Enzymol.

Methods Enzymol.

Anal. Biochem.

Methods Enzymol.

Anal. Biochem.

Anal. Biochem.

J. Biol. Chem.

Methods Enzymol.

Arch. Biochem. Biophys.

J. Biol. Chem.