Development of a rapid, highly sensitive, non-radioactive assay system for hematopoietic growth factors.
The aim of this study was to develop non-radioactive cell line proliferation assays. The human leukemic cell line TF1 (Kitamura et al., 1989) was used for the determination of the specific biological activity of recombinant human (rhu) granulocyte-macrophage colony-stimulating factor (GM-CSF) and rhu Interleukin 3 (IL-3) by a simple and economical fluorometric assay with a sensitivity similar to the measurement of 3H-thymidine uptake. The TF1 cell line responds to rhu IL-3, rhu GM-CSF and to a lesser extent to rhu Erythropoietin (EPO) and mast cell growth factor (MGF), but not to rhu G-CSF. It is dependent upon rhu GM-CSF for survival in culture. For the proliferation assay 1 x 10(4) TF1 cells were incubated with 20 ng - 0.256 pg rhu GM-CSF or rhu IL-3 at 37 degrees C and 5% CO2 in humidified atmosphere. After 48 h the cells were washed twice with PBS and were incubated with 4-Methylumbelliferyl-heptanoate for 60 min. Fluorescence was determined on a Titertek Fluoroskan II (Flow Lab.), and results were given as fluorescence units using a 355 nm excitation filter and a 480 nm emission filter. The developed assay showed an interassay variability lower than 15%. The sensitivity of the proliferation assays in the same range as the thymidine incorporation assays.
Hintz-Obertreis
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Research Laboratories of Behringwerke AG, Marburg, Germany.
Krumwieh
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Seiler
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D001681:Biological Assay / Q000379:methods; D002455:Cell Division / Q000187:drug effects*; D002460:Cell Line; D004261:DNA Replication / Q000187:drug effects; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000032:analysis* / Q000494:pharmacology; D006801:Humans; D007377:Interleukin-3 / Q000032:analysis* / Q000494:pharmacology; D007938:Leukemia; D011994:Recombinant Proteins / Q000032:analysis / Q000494:pharmacology; D013050:Spectrometry, Fluorescence / Q000379:methods; D013936:Thymidine / Q000032:analysis
D007377:Interleukin-3; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013936:Thymidine