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KMID : 0617220020130010113
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Duksung Bulletin Phamaceutical Sciences
2002 Volume.13 No. 1 p.113 ~ p.119
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In Vitro Bioassay for Transforming Growth Factor-βUsing XTT Method
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Kim, Mi-Sung
Ahn, Seong-Min/Moon, Aree
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Abstract
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Research in the cytokine field has grown exponentially in recent years, and the validity of such studies relies heavily on the appropriate measurement of levels of cytokines in various biological samples. Transforming growth factor (TGF)-β, a hormonally active polypeptide found in normal and transformed tissue, is a potent regulator of cell growth and differentiation. The most widely used bioassay for TGF-β, is the inhibition of the proliferation of mink lung epithelial cells. Though detection of [^3H]thymidine incorporation is more sensitive than the MTT assay, it presents some disadvantages due to the safety and disposal problems associated with radioisotopes. In this study, we attempted to ascrtain the experimental conditions which could be used for measuring the in vitro biological activity of TGF-βin a safer and more sensitive way compared with the currently available methods. We compared the commonly used method, the MTT assay, to the XTT assay using different parameters including cell number, incubation time and the wave length used for detecting the product. We examined the anti-proliferative activities of TGF-βin three different cell lines: Mv-1-Lu mink lung epithelial cells, MCF10A human breast epithelial cells and H-ras-transformed MCF10A cells. Herein, we present an experimental protocol which provides the most sensitive method of quantifying the biological activity of TGF-β, with a detection limit of as low as 10pg/ml: Mv-1Lu or H-ras MCF10A cells(1×10^5/well) were incubated with TGF-βat 37℃ in a humidified CO_2 incubator for 24 hr followed by XTT treatment and detemination of absorbance at 450 or 490 nm. Our results may contribute to the establishment of an in vitro bioassay system, which could be used for the satisfactory quantitation of TGF-β.
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