Fluorescence analysis of the labile iron pool of mammalian cells

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 salicyladehyde-isonicotinoyl-hydrazone (SIH) and the value of [CA]1; and (c) the "free" cell iron concentration [Fe(II)], which is computed from the experimentally determined values of CA-Fe(II)'s dissociation constant (Kd) in various cell lines grown in suspension (Kd = 0.22 +/- 0.01 microM). The value of cellular LIP is defined as the sum of [CA-Fe] and [Fe]. It is derived from the experimental determination of [CA]1 and [CA-Fe] and from calculation of [Fe] by application of the mass law equation using the Kd value of [CA-Fe]. The estimated values of LIP for resting erythroid and myeloid cells are in the range of 0.2-1.5 microM. The values varied commensurately with cell iron loads and iron chelator treatment. The method provides a simple, noninvasive tool for on-line monitoring of cytosolic iron under normal and abnormal conditions of cell iron supply and for assessing the dynamics of intracellular iron in living cells.