Quantitative colorimetric assay for basic fibroblast growth factor using bovine endothelial cells and heparin

Area of Science:

• Biochemistry
• Cell Biology
• Assay Development

Background:

• Basic fibroblast growth factor (bFGF) is crucial for cellular processes.
• Accurate quantification of bFGF is essential for research and diagnostics.
• Existing methods for bFGF detection can be complex or lack sensitivity.

Purpose of the Study:

• To establish a sensitive and reproducible colorimetric assay for quantifying basic fibroblast growth factor (bFGF) or bFGF-like activity.
• To optimize the assay using heparin to enhance bFGF detection limits and reduce variability.
• To validate the assay's performance against established methods.

Main Methods:

• Utilized a colorimetric assay based on the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), to measure bFGF-induced proliferation.
• Employed a bFGF-dependent endothelial cell line (ATCC CRL 1395).
• Investigated the effect of heparin on bFGF activity and assay variability.

Main Results:

• The MTT-based colorimetric assay accurately determined bFGF concentration.
• Addition of heparin (250 ng/mL) increased bFGF activity tenfold, enabling detection as low as 10 pg/mL.
• Heparin significantly reduced inter- and intra-assay variations.
• Assay results showed high correlation with [3H]-thymidine incorporation (r = 0.998) and cell counting (r = 0.996) assays.

Conclusions:

• A robust and reproducible colorimetric assay for bFGF quantification was developed.
• The assay is highly sensitive, especially when supplemented with heparin.
• This method is suitable for measuring bFGF or bFGF-like activity in various biological fluids and culture media.