Abstract
A simple and reproducible micro-agar culture technique for cloning human CFU-M is described. Human bone marrow mononuclear cells were suspended in agar and incubated for 12 days. Stimulation was provided by the direct addition of phytohemagglutinin-P (PHA-P), erythropoietin (Epo) and 2-mercaptoethanol (2-ME) to the liquid overlayer. A shift from BFU-E and CFU-C proliferation to CFU-M and CFU-TL was observed with increasing PHA concentrations. Under optimal conditions (PHA 50 micrograms, Epo 1.2 IU, 2-ME 2 x 10(-4) M, 1% purified BSA, 0.04% human transferrin, saturated with Fe C13) a linear relationship between colonies formed and plated cell number were observed. For the routine morphological analysis, the whole agar layers were stained using the Pappenheim method. For further characterization of CFU-M, cytochemical stainings and immunofluorescence tests with rabbit-antihuman factor VIII-related antigen were performed on the whole agar layers.
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