A simple and quantitative liquid culture system to measure megakaryocyte growth using highly purified CSU-MK

H. Miyazaki, K. Horie, Y. Shimada, A. Kokubo, E. Maeda, H. Inoue, Takashi Kato

Research output: Contribution to journalArticle

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Abstract

A new and quantitative liquid culture system has been developed to measure the production of megakaryocytes from megakaryocyte progenitor cells (colony-forming units-megakaryocyte [CFU-MK]). The system uses as a target population a glycoprotein (Gp) IIb/IIIa+ subpopulation of rat bone marrow cells previously demonstrated to be highly enriched for CFU-MK. GpIIb/IIIa+ cells were cultured at 5x104 cells/ml (104 cells/well) with test samples in 96-well tissue culture plates for 4 days at 37°C. During the final 3 hours of incubation, the cells were pulsed with [14C]5-hydroxytryptamine creatinine sulfate (14C-serotonin). After incubation, the plates were washed and the cell pellets were lysed with Triton-X 100. The cell lysate was infiltrated into a commercially available solid scintillator and dried, and radioactivity was measured. In this assay system, rat interleukin-3 (IL-3) was found to be the most potent among known cytokines tested. Murine granulocyte-macrophage colony-stimulating factor (GM-CSF), human erythropoietin (Epo), human IL-6, and murine stem cell factor (SCF) each alone stimulated megakaryocyte growth but were much less active than rat IL-3. Plasma of rats rendered thrombocytopenic by injection of monoclonal antirat platelet GpIIb/IIIa antibody exhibited significant activity, and the active protein fractions partially purified from the plasma showed much higher activity, but normal rat plasma had no effect. This liquid culture system allows the measurement of a large number of test samples-including a wide variety of cytokines and unknown growth factors, alone or in combinations-and provides a simple method for evaluating the early proliferative events involving CFU-MK in the megakaryocyte differentiation pathway.

Original languageEnglish
Pages (from-to)1224-1228
Number of pages5
JournalExperimental Hematology
Volume23
Issue number11
Publication statusPublished - 1995
Externally publishedYes

Fingerprint

Megakaryocytes
Growth
Stem Cells
Interleukin-3
Megakaryocyte Progenitor Cells
Cytokines
Platelet Glycoprotein GPIIb-IIIa Complex
Stem Cell Factor
Health Services Needs and Demand
Octoxynol
Granulocyte-Macrophage Colony-Stimulating Factor
Erythropoietin
Bone Marrow Cells
Radioactivity
Cultured Cells
Interleukin-6
Intercellular Signaling Peptides and Proteins
Blood Platelets
Injections
Antibodies

Keywords

  • CFU-MK
  • Liquid culture
  • Megakaryocyte-GpIIb/IIIa
  • Serotonin incorporation

ASJC Scopus subject areas

  • Cancer Research
  • Cell Biology
  • Genetics
  • Hematology
  • Oncology
  • Transplantation

Cite this

Miyazaki, H., Horie, K., Shimada, Y., Kokubo, A., Maeda, E., Inoue, H., & Kato, T. (1995). A simple and quantitative liquid culture system to measure megakaryocyte growth using highly purified CSU-MK. Experimental Hematology, 23(11), 1224-1228.

A simple and quantitative liquid culture system to measure megakaryocyte growth using highly purified CSU-MK. / Miyazaki, H.; Horie, K.; Shimada, Y.; Kokubo, A.; Maeda, E.; Inoue, H.; Kato, Takashi.

In: Experimental Hematology, Vol. 23, No. 11, 1995, p. 1224-1228.

Research output: Contribution to journalArticle

Miyazaki, H, Horie, K, Shimada, Y, Kokubo, A, Maeda, E, Inoue, H & Kato, T 1995, 'A simple and quantitative liquid culture system to measure megakaryocyte growth using highly purified CSU-MK', Experimental Hematology, vol. 23, no. 11, pp. 1224-1228.
Miyazaki H, Horie K, Shimada Y, Kokubo A, Maeda E, Inoue H et al. A simple and quantitative liquid culture system to measure megakaryocyte growth using highly purified CSU-MK. Experimental Hematology. 1995;23(11):1224-1228.
Miyazaki, H. ; Horie, K. ; Shimada, Y. ; Kokubo, A. ; Maeda, E. ; Inoue, H. ; Kato, Takashi. / A simple and quantitative liquid culture system to measure megakaryocyte growth using highly purified CSU-MK. In: Experimental Hematology. 1995 ; Vol. 23, No. 11. pp. 1224-1228.
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