title: Quantitative colorimetric assay for basic fibroblast growth factor using bovine endothelial cells and heparin.
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.
authors.affiliation: Research and Development Division, Takeda Chemical Industries, Ltd., Osaka, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Kajio
authors.forename: K
authors.initials: K
authors.lastname: Kawahara
authors.forename: K
authors.initials: K
authors.lastname: Kato
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003124:Colorimetry / Q000379:methods; D003470:Culture Media / Q000737:chemistry; D004727:Endothelium / Q000737:chemistry / Q000166:cytology; D016222:Fibroblast Growth Factor 2 / Q000032:analysis*; D006493:Heparin / Q000494:pharmacology; D009206:Myocardium / Q000737:chemistry / Q000166:cytology; D013778:Tetrazolium Salts; D013844:Thiazoles
chemical_list: D003470:Culture Media; D013778:Tetrazolium Salts; D013844:Thiazoles; D016222:Fibroblast Growth Factor 2; D006493:Heparin; C022616:thiazolyl blue