Flow cytometry measurement of the labile iron pool in human hematopoietic cells

Abstract

Iron is important for many biological processes, and its deficiency or excess is involved in pathological conditions. Although most iron is firmly bound (e.g., in hemoglobin), some, the labile iron pool (LIP), is bound to low-affinity ligands. The level of LIP is regulated to meet the cell's requirements for iron but prevent excess. We describe herein a multiparameter flow cytometry procedure for measuring LIP in various human hematopoietic cells. Peripheral blood and bone marrow (BM) cells were loaded with calcein-AM, washed, and then incubated with or without the high-affinity iron-chelator Deferiprone (L1). Specific cell subpopulations were identified based on side-light scattering and expression of surface antigens. LIP was determined based on the ability of L1 to bind and remove iron from calcein and thereby increase the fluorescence emitted by the cells. Blood cells differ in their LIP content in the order monocytes > PMN > RBC > lymphocytes. Analysis of BM cells indicated a similar tendency among precursors of the different lineages. The results also showed that among myeloid precursors, LIP increases along cell maturation. Flow cytometry might be useful for evaluating LIP in various diseases and for studying the efficacy of iron-chelators.