Fibroblast growth factor basic bfgf

We assessed the ability of B16-F10^WT, B16-F10^CT and C10 cells to produce melanospheres, by suspending the cells (1 × 10³ cells per well) in ultralow attachment six-well plates (Fisher Scientific, Paris, France), in a serum-free medium (SFM) consisting of DMEM/F-12 supplemented with 20 ng/mL EGF (epidermal growth factor; PeproTech, Germany), 20 ng/mL bFGF (basic fibroblast growth factor; PeproTech, Germany), and B27 (1X) (Invitrogen, USA). In all experiments, cells were incubated at 37°C under a humidified atmosphere containing 5% CO₂, for up to seven days. The total number of melanospheres generated was compared between groups.

The human neural stem cell line ReNcell VM was kindly provided by Dr. A. Cuadrado [24 (link)]. ReNcell VM cells were cultured on Corning Matrigel hESC-Qualified Matrix (Corning, Corning, NY, USA)-coated plates with proliferation medium: neurobasal medium (Gibco, Waltham, MA, USA) supplemented with 2% (v/v) B27 supplement (Gibco), 2 mM glutamax (Gibco), 50 μg/mL gentamicin, 20 ng/mL b-FGF (Basic Fibroblast Growth Factor; Peprotech, Waltham, MA, USA), and 20 ng/mL EGF (Epidermal Growth Factor; Peprotech), as described in [24 (link)]. Cells were cultured at 37 °C in a 5% CO₂ atmosphere, and cell passages were performed every 3–4 days.
At 80–90% confluence, differentiation was induced by growth factor withdrawal from the proliferation medium and monitored using phase-contrast microscopy and expression analysis of neuronal precursor (Nestin, SOX-2, and Ki-67 (MKI67)), neuronal (β-tubulin III, synapsin I (SYN1), and synaptophysin (SYP)), dopaminergic (tyrosine hydroxylase (TH) and dopamine decarboxylase (AADC)), and glial markers (glial fibrillary acidic protein (GFAP) and oligodendrocyte transcription factor (OLIG2)). Meanwhile, the differentiation medium was changed every 2–3 days.

Spheres were generated and expanded in CSCs media composed of: advanced DMEM:F12 (GIBCO) supplemented with 1 × glutaMAX (GIBCO), 1 × B-27 (GIBCO), 1 × N2 (GIBCO), 20 ng/ml bFGF (basic fibroblast growth factor) (Invitrogen) and 50 ng/ml EGF (epidermal growth factor) (Peprotech). Five hundred cells per 500 µl of sphere medium were seeded in 24-well ultra-low attachment plates (Corning) as described previously [47 ]. After 7 days of incubation, spheres were typically >75 µm large. For serial passaging, 7-day spheres were harvested using 40 µm cell strainers, dissociated to single cells with trypsin, and then regrown for another 7 days. Cultures were kept no longer than 4 weeks after recovery from frozen stocks (passage 3–4).

Skin samples from the back of 8-week-old C57BL/6 female mice were carefully and separately dissected free from other tissue, placed in Hank's balanced salt solution (HBSS), and cut into approximately 1 mm² pieces using dissecting scissors. Then, the segments were digested in 0.25% trypsin/EDTA at 37°C for 45 min. The resulting cell suspensions were seeded and cultured in a six-well plate in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) medium (Gibco, Invitrogen) containing 15% embryonic stem cell screened FBS (ES-FBS; Gibco), 1% glutamine (Gibco), 1% penicillin–streptomycin (Gibco) and fibroblast growth factor-basic (bFGF) (Invitrogen, 4 ng/ml) at 37°C in 5% CO₂ in humidified air. Cells were passaged every 4–6 days.