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

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 KMnO(4) 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.