Isolation and Primary Cell Culture of Mouse Dorsal Root Ganglion Neurons

Materials and Reagents

1. T25 cell culture flask (25 cm²) (Greiner Bio-One GmbH, catalog number: 690160 )
   
2. 24-well plate (Greiner Bio-One GmbH, catalog number: 662160 )
   
3. Cover glasses (Marienfeld-Superior, catalog number: 0111550 )
   
4. Glass Pasteur pipettes (Marienfeld-Superior, catalog number: 3233050 )
   
5. Filtropur S 0.2 syringe filters (Sarstedt AG, catalog number: 83.1826.001 )
   
6. Syringe Omnifix^® (B. Braun Medical, catalog number: 4616103V )
   
7. Dissection dish (petri dish) (94 x 16 mm) (Sigma-Aldrich, catalog number: Z617636 ) with silicone pad
   
8. Greiner Petri dishes (35 x 10 mm) (Sigma-Aldrich, catalog number: P5112 )
   
9. 15 ml Cellstar centrifugation tubes (Greiner Bio-One GmbH, catalog number: 188261 )
   
10. Mice (2 to 6 months) (strain: C57BL/6)
    
11. Poly-L-lysine hydrobromide (Sigma-Aldrich, catalog number: P2636 )
    
12. HEPES (AppliChem GmbH, catalog number: A3268 )
    
13. H₃BO₃ (Carl Roth GmbH + Co., catalog number: 6943.1 )
    
14. Sodium tetraborate (Na₂B₄O₇) (Sigma-Aldrich, catalog number: 221732 )
    
15. Bovine Serum Albumin (PAA, catalog number: K41-001 )
    
16. HBSS (Thermo Fisher Scientific, Gibco^TM, catalog number: 14175-053 )
    
17. Neurobasal^®-A medium (Thermo Fisher Scientific, Gibco^TM, catalog number: 10888022 )
    
18. B-27^® Supplement (50x) (Thermo Fisher Scientific, Gibco^TM, catalog number: 17504-044 )
    
19. L-glutamine (200 mM) (Thermo Fisher Scientific, Gibco^TM, catalog number: 25030 )
    
20. Penicillin/streptomycin (100x) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140 )
    
21. Minimum Essential Medium (MEM) (Thermo Fisher Scientific, Gibco^TM, catalog number: 31095-029 )
    
22. D(+)-Glucose (C₆H₁₂O₆) (Merck Millipore Corporation, catalog number: 108337 )
    
23. Collagenase-II (Worthington Biochemical Corporation, catalog number: LS004176 )
    
24. Horse serum (Thermo Fisher Scientific, Gibco^TM, catalog number: 26050-88 )
    
25. Trypsin from bovine pancreas (Sigma-Aldrich, catalog number: T1426 )
    
26. β-nerve growth factor (β-NGF) (Preprotec, catalog number: 450-01 )
    
27. Borate buffer (see Recipes)
    
28. Poly-L-lysine hydrobromide (PLL) stock solution (see Recipes)
    
29. Poly-L-lysine hydrobromide (PLL) working solution (see Recipes)
    
30. Dissociation solution (see Recipes)
    
31. 30% Glucose stock solution (see Recipes)
    
32. 0.5% BSA-blocking solution (see Recipes)
    
33. DRG neuronal culture medium (see Recipes)
    
34. DRG preparation medium (see Recipes)
    
35. Collagenase-II solution (see Recipes)
    
36. 2% trypsin stock solution (see Recipes)
    

Equipment

1. CO₂ incubator (37 °C, 5% CO₂ concentration and 95% relative humidity) (Labotect, model: C-200 )
   
2. Laminar flow work bench (Heraeus Holding)
   
3. Stereo microscope (Carl Zeiss, model: Stemi 2000C )
   
4. Cold light source (equipped with a flexible dual branch light guide) (Carl Zeiss, model: CL1500ECO )
   
5. Scissors
   
   1. Standard scissors (Fine Science Tools, catalog number: 14002-12 )
      
   2. Student spring scissors (Fine Science Tools, catalog number: 91500-09 )
      
   3. Angled spring scissors (Fine Science Tools, catalog number: 15006-09 )
6. Forceps
   
   1. Dumont #7b medical forceps (Fine Science Tools, catalog number: 11270-20 )
      
   2. Dumont #4 forceps (Fine Science Tools, catalog number: 11242-40 )
      
   3. Dumont #5 mirror finish forceps (Fine Science Tools, catalog number: 11252-23 )
7. Bunsen burner Fireboy plus (Integra Biosciences)
   
8. Glass Pasteur pipettes (Marienfeld-Superior, catalog number: 3233050 )
   
9. Dropper bulb for Pasteur pipettes (Thermo Fisher Scientific, catalog number: 03-448-25 )
   
10. Centrifuge 5810R (Eppendorf AG)
    
11. Pipetman P20/P200/P1000 (Gilson)
    

Procedure