PMID: 9177722May 15, 1997Paper

Fluorescence analysis of the labile iron pool of mammalian cells

Analytical Biochemistry
S EpsztejnI Z Cabantchik

Abstract

The labile iron pool (LIP) of cells constitutes a cytosolic fraction of iron which is accessible to permeant chelators and contains the cells' metabolically and catalytically reactive iron. LIP is maintained by a balanced movement of iron from extra- and intracellular sources. We describe here an approach for tracing LIP levels in living cells based on the fluorescent probe calcein (CA). This probe binds Fe(II) rapidly, stoichiometrically, and reversibly while forming fluorescence-quenched CA-Fe complexes. Cells are loaded with CA via its acetomethoxy precursor CA-AM, attaining 1-10 microM intracellular concentrations and retaining full viability. LIP is defined here operationally as the sum of "free" and CA-bound iron of the cell. The method for assessing LIP is based on the measurement of: (a) the total intracellular concentration of CA in CA-loaded cells ([CA]1), which is estimated from fluorimetric measurements of CA in a given suspension of cells, the number of cells, and the cell volume; (b) the intracellular [CA-Fe], the concentration of [CA] bound to metals (> 95% iron), which is assessed from the relative rise in fluorescence (delta F) elicited by addition of highly permeant and high-affinity binding chelators such as ...Continue Reading

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Citations

Jun 26, 2012·Biochemistry·Nema D JhurryPaul A Lindahl
Feb 8, 2011·Journal of the American Chemical Society·Samuel S TanEric T Kool
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