Diaminofluorene stain detects erythroid differentiation in immature haemopoietic cells treated with EPO, IL-3, SCF, TGFbeta1, MIP-1alpha and IFNgamma.
We have combined in vitro clonogenic culture and a highly sensitive stain for haemoglobin to compare the influence of EPO, IL-3, SCF, TGFbeta1, MIP-1alpha and IFNgamma, 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 TGFbeta1 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.
McGuckin
Colin P
CP
King-George Laboratory, St George's Hospital Medical School, London, UK. c.mcguckin@kingston.ac.uk
Forraz
Nicolas
N
Liu
Wai M
WM
D002454:Cell Differentiation / Q000187:drug effects; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D004396:Coloring Agents; D015672:Erythroid Precursor Cells / Q000166:cytology* / Q000187:drug effects; D004921:Erythropoietin / Q000494:pharmacology; D005449:Fluorenes; D006133:Growth Substances / Q000494:pharmacology*; D006454:Hemoglobins / Q000032:analysis; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007377:Interleukin-3 / Q000494:pharmacology; D020014:K562 Cells; D019402:Macrophage Inflammatory Proteins / Q000494:pharmacology; D019089:Stem Cell Factor / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053773:Transforming Growth Factor beta1; D014407:Tumor Cells, Cultured; D020298:U937 Cells
D054405:Chemokine CCL3; D054407:Chemokine CCL4; D004396:Coloring Agents; D005449:Fluorenes; D006133:Growth Substances; D006454:Hemoglobins; D007377:Interleukin-3; D019402:Macrophage Inflammatory Proteins; D019089:Stem Cell Factor; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D004921:Erythropoietin; D007371:Interferon-gamma; C012978:2,7-fluorenediamine