Active Fibroblast Growth Factor 2, Basic (FGF2)

Basic fibroblast growth factor (FGF2), also known as bFGF, FGF-β is a member of a large family of structurally related heparin-binding proteins (the FGFs) involved in the regulation of cell proliferation, growth and differentiation. It involved in many biological processes including angiogenesis, embryonic development and wound healing. Additionally, FGF2 is a critical component of human embryonic stem cell culture medium. Besides, Caspase 1 (CASP1) has been identified as an interactor of FGF2, thus a binding ELISA assay was conducted to detect the interaction of recombinant mouse FGF2 and recombinant mouse CASP1. Briefly, FGF2 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100ul were then transferred to CASP1-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-FGF2 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution , wells were incubated 15-25 minutes at 37℃. Finally, add 50µL stop solution to the wells and read at 450nm immediately. The binding activity of FGF2 and CASP1 was shown in Figure 1, and this effect was in a dose dependent manner.

To test the effect of rmFGF2 on cell proliferation of 3T3 fibroblasts , Balb/c 3T3 cells were seeded into triplicate wells of 96-well plates at a density of 8, 000 cells/well and allowed to attach overnight, then the medium was replaced with 0.4% FBS standard DMEM prior to the addition of various concentrations of recombinant mouse FGF2. After incubated for 48h, cells were observed by inverted microscope and cell proliferation was measured by Cell Counting Kit-8 (CCK-8). Briefly, 10 µl of CCK-8 solution was added to each well of the plate，then measure the absorbance at 450 nm using a microplate reader after incubating the plate for 1-4 hours at 37 °C. Cell proliferation of Balb/c 3T3 cells after incubation with rhEGF for 48h observed by inverted microscope was shown in Figure 2. The dose-effect curve of rmFGF2 was shown in Figure 3. It was obvious that rmFGF2 significantly promoted cell proliferation of 3T3 cells. The ED50 for this effect is typically 0.2 ug/ml.