losartan-potassium and 2-7-fluorenediamine

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.

Topics: Cell Differentiation; Chemokine CCL3; Chemokine CCL4; Coloring Agents; Erythroid Precursor Cells; Erythropoietin; Fluorenes; Growth Substances; Hemoglobins; Humans; Interferon-gamma; Interleukin-3; K562 Cells; Macrophage Inflammatory Proteins; Stem Cell Factor; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; U937 Cells

We have developed a new colorimetric method to detect early erythroid clonogenic progenitor cells by virtue of their hemoglobin-containing progeny in vitro. The method relies upon the specific oxidation of 2,7-diaminofluorene (DAF) by the pseudoperoxidase activity of hemoglobin (Hb). Generation of fluorene blue (FB), the chromophore generated upon oxidation of DAF, was a linear function of Hb and erythrocyte concentration. Measurement of FB absorbance at 610 nm was 80 times more sensitive than measurement of Hb absorbance at 530 nm. The limit of resolution was approximately 8 pmol of Hb corresponding to the Hb content of approximately 5000 murine peripheral blood erythrocytes. FB concentration closely paralleled eight-day BFU-E colony formation as a function of erythropoietin dose. However, the method was unable to detect a response for CFU-E to erythropoietin because of persistently high background levels of Hb in two-day cultures. Generation of FB by cells likely to contain myeloperoxidase was negligible, and FB production was not correlated with the number of CFU-GM colonies. DAF oxidation by hemoglobin showed a higher hydrogen peroxide optimum than oxidation by microperoxidase, so that, under the conditions used, FB generation by peroxidase was not favored. As a further confirmation of specificity, peritoneal exudate cells failed to show significant generation of FB. The DAF reagent was also used to stain both CFU-E and BFU-E colonies in situ, and was more sensitive than benzidine dihydrochloride in the histochemical detection of Hb. DAF can replace hazardous benzidine-related compounds for the staining erythroid colonies in culture dishes, and the method offers a new quantitative approach to the study of erythropoietin responsiveness in vitro.

Topics: Animals; Cell Differentiation; Cells, Cultured; Colony-Forming Units Assay; Colorimetry; Erythropoiesis; Erythropoietin; Female; Fluorenes; Hemin; Hemoglobins; Interleukin-3; Iron; Mice; Peroxidases