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.
Rh bfgf
Manufactured by R&D Systems
Recombinant human basic Fibroblast Growth Factor (Rh bFGF) is a 17 kDa heparin-binding protein that belongs to the FGF family. It is a potent mitogen for a variety of cell types, including fibroblasts, myoblasts, osteoblasts, and endothelial cells.
Automatically generated - may contain errors
Lab products found in correlation
Heparin, by Merck Group (3 mentions)
Rhegf, by R&D Systems (3 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Dispase 2 protease, by Merck Group (2 mentions)
Collagenase type 1, by Worthington (1 mentions)
70 μm strainer, by BD (1 mentions)
Dispase protease type 2, by Cambrex (1 mentions)
3 protocols using rh bfgf
1
Culturing Embryonic Mouse Neurofibroma Spheres
2
Mouse Neurofibroma-Derived Sphere Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
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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.
3
Mouse Neurofibroma-Derived Sphere Culture
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