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.