Basic fibroblast growth factor (bfgf)

Snap-frozen tumor samples were crushed into fragments <2 mm in diameter and resuspended with 1:2 weight/volume of 2×PBS and rotated at 4°C for 2 h. The samples were vortexed then spun for 3 min at 5,000 RPM then the tumor extracellular fluid was collected36 (link). Collected cultured cells and diluted to 2 × 10⁶/ml with PBS. Broke up cells by ultrasonic and spun for 10 min at 10000 g then the cell lysate was collected. Serum samples and/or tumor extracellular fluid and/or cell lysate from the control and SH-treated groups were applied to ELISA kits, following the manufacturer 's protocol to quantify bFGF (CUSABIO, China), PF-4 (CUSABIO, China), G-CSF (RayBiotech, USA) and GM-CSF (RayBiotech, USA).

The dCD34⁺ cells and BMSCs were harvested and suspended in endothelial differentiation medium containing DMEM/F12 (HyClone), 10% FBS (ScienCell), 50 ng/mL VEGF (Abcam), 10 ng/mL bFGF (Abcam), 100 ng/mL endothelial cell growth supplement (ECGS; ScienCell), 1 ng/mL interleukin‐3 (IL‐3; PeproTech), 50 ng/mL interleukin‐11 (IL‐11; PeproTech) and 4.5 × 10⁻⁴ 1‐thioglycerol (Sigma). Cultures were incubated for 15 days. To detect endothelial function, cells (5.0 × 10⁴) were seeded in a 48‐well cell culture dish (Nest) coated with Matrigel matrix (BD). Endothelial differentiation was evaluated by indirect immunofluorescence staining for the expression of CD31 (Abcam) and vWF (Abcam).
For enzyme‐linked immunosorbent assay (ELISA), the supernatant of dCD34⁺ cells and BMSCs were collected after 24 hours of culture and analysed for VEGF and bFGF by ELISA kits (CUSABIO, Wuhan, Hubei, China) according to the manufacturer's protocol. Basal media were also measured as negative control.