Simultaneous assay for megakaryocyte colony-stimulating factor and megakaryocyte potentiator and its application

We have devised a simultaneous assay system for megakaryocyte colony-stimulating factor (Meg-CSF) and megakaryocyte potentiator (Meg-Pot) by modifying a quantitative measuring technique for acetylcholinesterase activity (Ach-E) of megakaryocytes by automatic colorimetry using microplates. We cultured murine bone marrow cells treated with diisopropyl fluorophosphate in a serum-free system with serum-free pokeweek mitogen-stimulated spleen cell conditioned medium (PWM-SCM) and an unknown factor, preparing two microplates with the identical culture system. In the first plate, the total number of Ach-E-positive cells induced solely by the factor tested was indicative of Meg-CSF activity and additive increases in this parameter on simultaneous addition of PWM-SCM and the factor tested were indicative of early Meg-Pot activity. Total Ach-E activity (total change at optical density of 414 nm) per well was measured in the second plate to calculate total change at optical density of 414 nm per megakaryocyte, an indicator of late Meg-Pot activity. With this system, recombinant human erythropoietin showed both Meg-CSF and early and late Meg-Pot activities in in vitro megakaryopoiesis. Recombinant murine granulocyte-macrophage colony-stimulating factor possessed weak Meg-CSF and early Meg-Pot activity, whereas recombinant human granulocyte colony-stimulating factor exhibited late Meg-Pot activity and thrombocytopenic serum exhibited early and late Meg-Pot activities. This assay system is useful in screening Meg-CSF or Meg-Pot activities in unknown factors.