Serotonin incorporation as a marker of murine megakaryocyte proliferation in liquid cultures.
We have developed a new in vitro method for the quantitation of murine megakaryocyte proliferation that is based on the unique property of megakaryocytes to incorporate and store [14C]serotonin in cytoplasmic dense granules. The specificity of the assay was demonstrated by autoradiography of whole bone marrow cell suspensions, which showed evidence of grain accumulation only in megakaryocytes. Bone marrow cells were cultured in liquid cultures in the presence of a stimulator of megakaryocyte growth before the addition of 2.5 microM [14C]serotonin. The amount of serotonin incorporated in cells was evaluated after 3 h. Radioactivity peaked at days 6 and 7 and remained high until day 10; there was a linear relationship between the incorporation of serotonin and the number of cells plated. A dose-response curve between the incorporation of serotonin and the concentration of pokeweed mitogen spleen-conditioned medium (PWM-SCM) was observed, with inhibitory effects becoming predominant at the highest concentrations. The proliferation of megakaryocyte progenitors was also stimulated by partially purified interleukin 3, whereas both human recombinant erythropoietin and human recombinant granulocyte colony-stimulating factor (rG-CSF) failed to modify the incorporation of serotonin in comparison with unstimulated cultures. Finally, in parallel experiments we observed a significant correlation between the number of megakaryocytic colonies grown in agar and the radioactivity in liquid cultures. The method described herein is reproducible, sensitive, and easy to perform; it should be useful for the study and purification of factors affecting megakaryocyte proliferation.
Vannucchi
A M
AM
Department of Hematology, University of Florence, Italy.
Grossi
A
A
Rafanelli
D
D
Vannucchi
L
L
Ferrini
P R
PR
D000818:Animals; D001854:Bone Marrow Cells; D002250:Carbon Radioisotopes / Q000378:metabolism; D002455:Cell Division; D003114:Colony-Forming Units Assay; D003470:Culture Media; D008533:Megakaryocytes / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D011043:Pokeweed Mitogens / Q000494:pharmacology; D012701:Serotonin / Q000378:metabolism*; D013997:Time Factors
D002250:Carbon Radioisotopes; D003470:Culture Media; D011043:Pokeweed Mitogens; D012701:Serotonin