FLUORESCENCE ANALYSIS OF THE LABILE IRON POOL OF MAMMALIAN-CELLS

The labile iron pool (LIP) of cells constitutes a cytosolic fraction o f iron which is accessible to permeant chelators and contains the cell s' 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 base d on the fluorescent probe calcein (CA). This probe binds Fe(II) rapid ly, stoichiometrically, and reversibly while forming fluorescence-quen ched CA-Fe complexes, Cells are loaded with CA via its acetomethoxy pr ecursor CA-AM, attaining 1-10 mu M intracellular concentrations and re taining full viability, LIP is defined here operationally as the sum o f ''free'' and CA-bound iron of the cell, The method for assessing LIP is based on the measurement of: (a) the total intracellular concentra tion of CA in CA-loaded cells ([CA](t)), which is estimated hom fluori metric measurements of CA in a given suspension of cells, the number o f cells, and the cell volume; (b) the intracellular [CA-Fe], the conce ntration of [CA] bound to metals (>95% iron), which is assessed from t he relative rise in fluorescence (Delta F) elicited by addition of hig hly permeant and high-affinity binding chelators such as salicyladehyd e-isonicotinoyl-hydrazone (SIH) and the value of [CA](t); and (c) the ''free'' cell iron concentration [Fe(II)], which is computed from the experimentally determined values of CA-Fe(II)'s dissociation constant (K-d) in various cell lines grown in suspension (K-d = 0.22 +/- 0.01 m u M). The value of cellular LIP is defined as the sum of [CA-Fe] and [ Fe]. It is derived from the experimental determination of [CA](t) and [CA-Fe] and from calculation of [Fe] by application of the mass law eq uation using the K-d value of [CA-Fe], The estimated values of LIP for resting erythroid and myeloid cells are in the range of 0.2-1.5 mu M. The values varied commensurately with cell iron loads and iron chelat or treatment, The method provides a simple, noninvasive tool for on-li ne monitoring of cytosolic iron under normal and abnormal conditions o f cell iron supply and for assessing the dynamics of intracellular iro n in living cells. (C) 1997 Academic Press.