Mouse Neurofibroma-Derived Sphere Culture

Mouse neurofibroma/DRG-derived sphere culture was performed as described (39 (link)). We dissected mouse tumors from DRG/tumors and cut them into 1 mm³ pieces in L-15 media supplemented with Pen/Strep (Thermo Fisher Scientifics, Cat# 15-140-122), Collagenase Type I (0.5mg/mL, Worthington Boichemical, Lakewood, NJ, Cat # LS004196), and Dispase protease II (2.5mg/mL, Sigma-Aldrich, Burlington, MA, Cat # 04942078001). We dissociated the tumors for 4 hours at 37°C while shaking at 170 RPM. We then passed the dissociated cells through a 40mm cell strainer. We plated the trypan blue negative cells at 1 × 10⁴ cells/well in 24-well low-binding plates in 1mL sphere medium containing DMEM:F-12 (3:1) + 20 ng/ml rhEGF (R&D Systems, Minneapolis, MN, Cat # 236-EG-200), 20 ng/ml rh bFGF (R&D Systems, Cat# 233-FB-025 ), 1% B-27 (Thermo Fisher Scientifics, Cat# 17504–044), and 2 μg/ml heparin (Sigma-Aldrich, St Louis, MO, Cat# H3149 ) at 37°C and 5% CO₂. We added 0.25 mL sphere medium to each well twice a week. We used secondary spheres for all experiments.