Diaminofluorene stain detects erythroid differentiation in immature haemopoietic cells treated with EPO, IL-3, SCF, TGFβ1, MIP-1α and IFNγ
Colin P. McGuckin,
Nicolas Forraz,
Wai M. Liu,
Colin P. McGuckin
King-George Laboratory, St George's Hospital Medical School and Kingston University, London, UK;
School of Life Sciences, Kingston University, Kingston upon Thames, UK;
Search for more papers by this authorNicolas Forraz
King-George Laboratory, St George's Hospital Medical School and Kingston University, London, UK;
School of Life Sciences, Kingston University, Kingston upon Thames, UK;
Department of Haematology, St George's Hospital Medical School, London, UK
Search for more papers by this authorWai M. Liu
Department of Haematology, St George's Hospital Medical School, London, UK
Search for more papers by this authorColin P. McGuckin,
Nicolas Forraz,
Wai M. Liu,
Colin P. McGuckin
King-George Laboratory, St George's Hospital Medical School and Kingston University, London, UK;
School of Life Sciences, Kingston University, Kingston upon Thames, UK;
Search for more papers by this authorNicolas Forraz
King-George Laboratory, St George's Hospital Medical School and Kingston University, London, UK;
School of Life Sciences, Kingston University, Kingston upon Thames, UK;
Department of Haematology, St George's Hospital Medical School, London, UK
Search for more papers by this authorWai M. Liu
Department of Haematology, St George's Hospital Medical School, London, UK
Search for more papers by this authorColin P. McGuckin, PhD, School of Life Sciences, Faculty of Science, Kingston University, Penrhyn Road, Kingston Upon Thames, Surrey KT1 2EE, UK Tel: +44 797 126 6764 Fax: +44 208 547 7562 e-mail: [email protected]
Abstract
Abstract: We have combined in vitro clonogenic culture and a highly sensitive stain for haemoglobin to compare the influence of EPO, IL-3, SCF, TGFβ1, MIP-1α and IFNγ, to directly stimulate cells in the progenitor compartment to develop towards the erythroid lineage. Three cell lines were chosen, as they exist developmentally arrested in the progenitor compartment, yet in a pliant state of maturation. HEL (erythroleukaemia) and K562 (CML-derived) cell lines, may, under appropriate stimuli, develop erythroid characters, whilst the third, U937 (as control cell line), may be stimulated by DMSO to differentiate to myeloid cells. After in vitro semi-solid methylcellulose culture with these cytokines, resulting colonies were stained with 2,7-diaminofluorene (DAF), which sensitively stains haemoglobin blue. Haemoglobin production was low in HEL and K562 cells and absent in U937. Cytokine analysis showed varying levels of influence depending on the starting level of cell line maturation. EPO and TGFβ1 maximally stimulated haemoglobin production in the HEL and K562 cell lines. This differential cytokine stimulation analysis combined with sensitive DAF haemoglobin detection could be applied in the study of many erythropoiesis-deficient patients or primitive erythropoiesis.
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