EC50 (+Heparin): [3H]thymidine incorporation in response to the stimulation of NIH 3T3
Liquid scintillation counting
0.1 to 100 ng/mL
The mitogenic properties of FGF-1 mutants were assessed by monitoring [3H]thymidine incorporation in response to the stimulation of NIH 3T3 cells. The cells were starved in serum-free DMEM containing 2.5 μg/mL insulin and 2.5 μg/mL transferrin for 24 h at 37 °C. Then, increasing amounts (0.1 to 100 ng/mL) of FGF-1 or its mutants were added, and the incubation was continued for 24 h at 37 °C in the presence of 10 U/mL heparin. For the last 6 h, 1 μCi/mL [3H]-thymidine was added to the cells. Finally, the cells were treated with 5% trichloroacetic acid and then dissolved in 0.1 M NaOH, and the level of trichloacetic acid-insoluble radioactivity was measured using a liquid scintillation beta-counter. The relationship between the [3H]thymidine incorporation and log concentration of the added growth factor was fitted to a sigmoidal function. Specific mitogenic activity was expressed as the concentration giving 50% of maximal stimulation (EC50).