All work using animals conformed to UK Home Office legislation (Scientific Procedures Act 1986). Schwann cells were harvested from the sciatic nerve and brachial plexus of postnatal Day 3 or 4 rats. Rat Schwann cells are used as well established culture protocols exist and they can be successfully expanded several times in vitro in order to create cryopreserved stocks. Nerves were enzymatically digested with a collagenase (Sigma, 3 mg/ml) and dispase II protease (Sigma, 2.5 mg/ml) incubation for 1 h at 37°C. Nerves were gently triturated using a P1000 pipette tip and plated onto PDL/laminin coated plastic in Schwann cell expansion medium [DMEM/F12 (ThermoFisher), 10% foetal bovine serum (ThermoFisher), 200 ng/ml NRG1-β1 EGF domain (R&D Systems), 10 ng/ml NGF (recombinant-murine, Peprotech), 4 µg/ml forskolin (Sigma)]. Cells were serially treated with 5–10 μM araC to eliminate fibroblasts. After approximately four passages, each time doubling the growing area, cells were frozen in a Mr Frosty freezing container (ThermoFisher), and stored in liquid nitrogen.
Dispase 2 protease
Manufactured by Merck Group
Sourced in United States
Dispase II protease is a highly active, genetically engineered bacterial protease enzyme. It is effective in dissociating a variety of cell types, including epithelial, endothelial, and fibroblast cells, from their extracellular matrices. The enzyme functions by cleaving peptide bonds in the collagen and fibronectin components of the extracellular matrix.
Automatically generated - may contain errors
Lab products found in correlation
Pen strep, by Thermo Fisher Scientific (4 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Heparin, by Merck Group (2 mentions)
Rhegf, by R&D Systems (2 mentions)
Rh bfgf, by R&D Systems (2 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Collagenase, by Merck Group (1 mentions)
Forskolin, by Merck Group (1 mentions)
Mr frosty freezing container, by Thermo Fisher Scientific (1 mentions)
Keratinocyte sfm, by Thermo Fisher Scientific (1 mentions)
Collagenase type 1, by Harvard Bioscience (1 mentions)
Hanks balanced salt solution (hbss), by Merck Group (1 mentions)
Trypsin edta, by Wisent (1 mentions)
Hanks balanced salt solution (hbss), by Wisent (1 mentions)
5 protocols using dispase 2 protease
1
Rat Schwann Cell Expansion Protocol
2
Isolation of Epidermal Cell-Derived Extracellular Vesicles
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Fresh Mouse skin from animal surgery was taken, washed 2-3 times with DPBS containing 1% antibiotics. The epidermis and dermis were separated by incubating in 2 mg/mL Dispase II protease (Sigma, USA) overnight at 4℃, and then epidermis was cultured in Keratinocyte-SFM (Gibco, USA) at 37℃, in 5% carbon dioxide, and 95% relative humidity. Keratinocyte-SFM is a complete serum-free medium when supplemented with human recombinant Epidermal Growth Factor (EGF) and Bovine Pituitary Extract (BPE) at the time of use. After 24 h of culture, cell supernatants were collected and centrifuged to remove cell debris, after which were collected for EV isolation.
3
Dissection and Culture of Murine DRG Neurons
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DRGs from 8-12 week old male mice were dissected, dissociated and cultured as follows: freshly dissected DRGs were initially stored in Hank's Balanced Salt Solution (HBSS, Wisent, #311-511-CL), then enzymatically dissociated by incubating first with 0.25% trypsin-EDTA (Wisent, #325-043-EL) for 20 minutes at 37 C, then with collagenase type I (Biochrom, # C 1-28; 2 mg/ml) and dispase II protease (Sigma, #D4693; 5 mg/ml) in HBSS for 30-40 minutes at 37 C. The suspension was centrifuged for 2 minutes at 800 rpm, the enzymatic solution was removed and replaced with 200 ml of DRG culture media (for composition, see Hu and Lewin, 2006) . DRG neurons were mechanically separated by trituration and plated on poly-L-lysine-(100 mg/ml) and laminin-(20 mg/ml) coated 35 mm glass-bottom FluoroDishes (World Precision Instruments Inc., #FD3510-100) and incubated at 37 C.
4
Mouse Neurofibroma-Derived Sphere Culture
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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.
5
Mouse Neurofibroma-Derived Sphere Culture
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