Quantitative colorimetric assay for basic fibroblast growth factor using bovine endothelial cells and heparin. |
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Authors: | T Kajio K Kawahara K Kato |
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Affiliation: | Research and Development Division, Takeda Chemical Industries, Ltd., Osaka, Japan. |
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Abstract: | An accurate and reproducible colorimetric assay was established to determine the concentration of basic fibroblast growth factor (bFGF) or bFGF-like activity in culture media and biological fluids. Fetal bovine heart endothelial cell line ATCC CRL 1395 was used as the bFGF-dependent cell line. The proliferation-stimulating activity of bFGF was determined by measuring the amount of formazan formed by the mitochondrial enzymes from a tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), instead of counting the viable cell numbers or measuring the incorporation of [3H]-thymidine. The addition of 250 ng/mL of heparin to the culture medium resulted in about a tenfold increase in the proliferation-stimulating activity of bFGF and allowed the detection of as low as 10 pg/mL of bFGF. Heparin also resulted in much smaller inter- and intraassay variations. The bFGF concentrations determined by this colorimetric assay correlated well with those determined by both the [3H]-thymidine incorporation assay using BALB/c 3T3 fibroblast cells (r = 0.998) and the cell number count assay (r = 0.996). This assay can be adapted to quantify bFGF or bFGF-like activity in tissue culture media and biological fluids such as plasma and organ extracts. |
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