Recombinant human fgf2 100 18c

First, primary cell populations isolated from fetal adrenal glands were cultured in 6-well plates (3516, Corning) with 2 ml of medium in RPMI 1640 with 10% FBS (SH30070.03, HyClone), 1% penicillin/streptomycin (15140-122, Gibco) and 2 mM L-glutamine (25030081, Gibco) at 37°C in a humidified 5% CO₂ atmosphere. After 7 days, expanded adherent cells were trypsinized and reseeded in DMEM/F12 supplemented with 10% FBS, 5% horse serum (16050-122, Gibco), 100 μg/ml primocin (ant-pm-1, In vivoGen), 100 ng/ml recombinant human FGF2 (100-18C, PeproTech), and 2 mM L-glutamine (28 (link)). Primary cell populations were stained with CYP17A1 and MC2R to confirm that they were steroidogenic cells.

Tumor cells (2−3 × 10³), endothelial cells (2 × 10³), and pericytes (1 × 10³) were seeded onto each well of a 96-well plate. Proliferation of tumor cells treated with indicated doses of VEGF blockade, PDGFRβ blockade, and a control vehicle was analyzed at 24, 48, 72, and 96 h using an MTT (5 mg mL⁻¹; M5655; Sigma-Aldrich) assay. Endothelial cells and pericytes were incubated with conditioned media collected from tumor cell culture diluted in fresh DMEM supplemented with 2% FBS. Cell proliferation was measured at 72 h. Recombinant human FGF-2- (100-18 C; PEPROTECH) treated endothelial cells (5 ng mL⁻¹) and pericytes (50 ng mL⁻¹) were used as positive controls. Absorbance values at 490 nm were obtained using a spectrophotometer (FLUOstar Omega; BMG LABTECH).