Dispase protease type 2

Embryonic mouse spheres, dissociated from E12.5 DRG with 0.25% Trypsin 20 min. at 37°C (Mediatech; Herndon, VA) produced single-cell suspensions with narrow-bore pipettes and a 70 μm strainer (BD-Falcon). For mouse or human neurofibroma spheres, we chopped tissue into 1–3 mm³ pieces, plated in 20mL L-15 (Mediatech) plus 0.5 mg/mL collagenase type 1 (Worthington; Lakewood, NJ), and 2.5 mg/mL dispase protease type II (Cambrex; East Rutherford, NJ) at 37°C for 4–6 hours. We plated trypan blue negative cells (Stem Cell Technologies, Vancouver, BC) at 1 × 10⁴ cells in 1 mL per well in 24-well low-binding plates in medium containing DMEM:F-12 (3:1) + 20 ng/ml rhEGF (R&D Systems), 20 ng/ml rh bFGF (R&D Systems), 1% B-27 (Invitrogen), 2 μg/ml heparin (Sigma). We maintained cultures at 37°C and 5% CO₂ and counted floating spheres after 4–7 days. To passage, we centrifuged sphere cultures, dissociated and plated at 1 × 10⁴ cells/ml in fresh sphere medium as described (Williams et al., 2008 (link)). For each experiment, we show a representative of 3 independent experiments.