Isolation and Primary Cell Culture of Mouse Dorsal Root Ganglion Neurons
小鼠背根神经节神经元的分离和原代细胞培养
发布: 2016年04月05日第6卷第7期 DOI: 10.21769/BioProtoc.1785 浏览次数: 32098
评审: Xuecai GeAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jun 2015
Abstract
We here provide a detailed protocol for the isolation and culture of primary mouse sensory neurons. The cell bodies of sensory afferent pseudounipolar neurons are located in dorsal root ganglia (DRGs) along the vertebral column. Dissected mouse DRGs can be dissociated into single cells by enzymatic digestion to obtain primary cultures of mouse sensory neurons as performed in the studies reported by Khaminets et al. (2015).
Materials and Reagents
- T25 cell culture flask (25 cm2) (Greiner Bio-One GmbH, catalog number: 690160 )
- 24-well plate (Greiner Bio-One GmbH, catalog number: 662160 )
- Cover glasses (Marienfeld-Superior, catalog number: 0111550 )
- Glass Pasteur pipettes (Marienfeld-Superior, catalog number: 3233050 )
- Filtropur S 0.2 syringe filters (Sarstedt AG, catalog number: 83.1826.001 )
- Syringe Omnifix® (B. Braun Medical, catalog number: 4616103V )
- Dissection dish (petri dish) (94 x 16 mm) (Sigma-Aldrich, catalog number: Z617636 ) with silicone pad
- Greiner Petri dishes (35 x 10 mm) (Sigma-Aldrich, catalog number: P5112 )
- 15 ml Cellstar centrifugation tubes (Greiner Bio-One GmbH, catalog number: 188261 )
- Mice (2 to 6 months) (strain: C57BL/6)
- Poly-L-lysine hydrobromide (Sigma-Aldrich, catalog number: P2636 )
- HEPES (AppliChem GmbH, catalog number: A3268 )
- H3BO3 (Carl Roth GmbH + Co., catalog number: 6943.1 )
- Sodium tetraborate (Na2B4O7) (Sigma-Aldrich, catalog number: 221732 )
- Bovine Serum Albumin (PAA, catalog number: K41-001 )
- HBSS (Thermo Fisher Scientific, GibcoTM, catalog number: 14175-053 )
- Neurobasal®-A medium (Thermo Fisher Scientific, GibcoTM, catalog number: 10888022 )
- B-27® Supplement (50x) (Thermo Fisher Scientific, GibcoTM, catalog number: 17504-044 )
- L-glutamine (200 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 25030 )
- Penicillin/streptomycin (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140 )
- Minimum Essential Medium (MEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 31095-029 )
- D(+)-Glucose (C6H12O6) (Merck Millipore Corporation, catalog number: 108337 )
- Collagenase-II (Worthington Biochemical Corporation, catalog number: LS004176 )
- Horse serum (Thermo Fisher Scientific, GibcoTM, catalog number: 26050-88 )
- Trypsin from bovine pancreas (Sigma-Aldrich, catalog number: T1426 )
- β-nerve growth factor (β-NGF) (Preprotec, catalog number: 450-01 )
- Borate buffer (see Recipes)
- Poly-L-lysine hydrobromide (PLL) stock solution (see Recipes)
- Poly-L-lysine hydrobromide (PLL) working solution (see Recipes)
- Dissociation solution (see Recipes)
- 30% Glucose stock solution (see Recipes)
- 0.5% BSA-blocking solution (see Recipes)
- DRG neuronal culture medium (see Recipes)
- DRG preparation medium (see Recipes)
- Collagenase-II solution (see Recipes)
- 2% trypsin stock solution (see Recipes)
Equipment
- CO2 incubator (37 °C, 5% CO2 concentration and 95% relative humidity) (Labotect, model: C-200 )
- Laminar flow work bench (Heraeus Holding)
- Stereo microscope (Carl Zeiss, model: Stemi 2000C )
- Cold light source (equipped with a flexible dual branch light guide) (Carl Zeiss, model: CL1500ECO )
- Scissors
- Forceps
- Bunsen burner Fireboy plus (Integra Biosciences)
- Glass Pasteur pipettes (Marienfeld-Superior, catalog number: 3233050 )
- Dropper bulb for Pasteur pipettes (Thermo Fisher Scientific, catalog number: 03-448-25 )
- Centrifuge 5810R (Eppendorf AG)
- Pipetman P20/P200/P1000 (Gilson)
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Heinrich, T., Hübner, C. A. and Kurth, I. (2016). Isolation and Primary Cell Culture of Mouse Dorsal Root Ganglion Neurons. Bio-protocol 6(7): e1785. DOI: 10.21769/BioProtoc.1785.
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分类
神经科学 > 感觉和运动系统 > 细胞分离和培养
细胞生物学 > 组织分析 > 组织分离
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