Recombinant fgf2

For inhibitor experiments, cells were treated for 1 h with FGFR inhibitor PD173074 (2 μM) before stimulation with 100 ng/ml recombinant FGF2 (PeproTech, London, UK) and 300 ng/ml heparin sodium salt (Sigma-Aldrich), for different time points. Similarly, conditioned media (serum-free) from normal or cancer cells were added.

The CT-2A-luc cells were generously donated by Dr. Martha R. Neagu. The cells were incubated at 37 °C with humidified air containing 5% CO₂. Monolayer CT-2A-luc cells were cultured in Dulbecco’s modified eagle medium with high glucose (DMEM; Thermo Fisher Scientific, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin-streptomycin. To generate neurospheres, CT-2A monolayer cells were enzymatically dissociated by accutase (Stem Cell Technology, San Diego, CA, USA) and seeded in 25 cm² culture dishes at the cell concentration of 1 × 10⁵ cells/mL in serum-free medium, composed of advanced DMEM/F12 medium (Life Technologies, Carlsbad, CA, USA) with l-glutamine (2 mM; Cellgro, Manassas, VA, USA), 1% N2 supplement (Life Technologies), 1% penicillin-streptomycin (Cellgro), recombinant EGF (20 ng/mL; R&D Systems, Minneapolis, MN, USA), and recombinant FGF2 (20 ng/mL; Peprotech, East Windsor, NJ, USA). After 10–11 days, the neurospheres were collected, enzymatically dissociated with accutase (Stem Cell Technology), and prepared for further studies [20 (link),21 (link)].

3F12E7 scFv binding to FGF2 was initially verified by sandwich ELISA. Briefly, 96-well plates were coated overnight at 4 °C with 5 µg/mL of (i) 3F12E7 scFv; (ii) 3F12E7 full-length IgG mAb (positive control); or (iii) irrelevant full-length IgG (negative control). The wells were blocked with 1% bovine serum albumin (BSA; Sigma, USA) in phosphate-buffered saline (PBS) and, then, incubated (or not) with 50 ng/mL recombinant FGF2 (PeproTech Inc., USA). Subsequently, wells were incubated with polyclonal anti-FGF2 antibody (1:10,000; Sigma, USA) and then with biotin-conjugated secondary antibody (1:5000; Sigma, USA), both diluted in 0.1% BSA containing 0.1% Tween-20 (Sigma, USA) (PBST).
The binding of size exclusion chromatographic elution fractions of 3F12E7 scFv to FGF2 was detected by direct ELISA. For that, wells were coated overnight at 4 °C with 3F12E7 scFv (5 µg/mL). After blocking with 1% BSA in PBS, wells were incubated with FGF2 (PeproTech Inc., USA) labeled to biotin. FGF2 biotinylation was performed using the EZ-Link Sulfo-NHS-Biotin kit (Thermo Scientific, USA).
In all cases, the wells were washed three times with PBST between each step. The reactions were revealed with horseradish peroxidase (HRP)-streptavidin (1:2000; Sigma, USA) and o-phenylenediamine (OPD; Sigma, USA) substrate. Absorbance values were read at 490 nm.