Materials and Reagents

1. Pipette tips
   
2. Coverglasses (Thermo Fisher Scientific or VWR)
   
3. Falcon tubes (50 ml)
   
4. Falcon tubes (15 ml)
   
5. Sterile pipettes 5, 10, 25 ml
   
6. 12-well culture plate
   
7. 10, 15 cm cell culture dishes
   
8. Razor blades
   
9. Fire-polished glass Pasteur pipettes
   

Note: Materials #3-9 can be from various suppliers.

10. Microfluidic chamber (XONA MICROFLUIDICS, catalog number: SND450 )
    
11. Stericup^® filter units (150, 250 ml) (EMD Millipore, catalog number: SCVPU02RE , 0.10 µm, polyethersulfone)
    
12. Pregnant female Sprague Dawley rats (Embryos dissected at E17.5) (Harlan Sprague Dawley)
    
13. Poly-L-lysine 0.1% (w/v) in dH₂O (Sigma-Aldrich, catalog number: P8920 )
    
14. Phosphate-buffered saline (PBS) (without CaCl₂, MgCl₂) (Thermo Fisher Scientific, Gibco^TM, catalog number: 1419094 )
    
15. 70% ethanol
    
16. 10x, 2.5% trypsin, no phenol red (Thermo Fisher Scientific, Gibco^TM, catalog number: 15090046 )
    
17. 10x DNase I (10 mg/ml) (Roche Diagnostics, catalog number: 10104159001 )
    
18. Anti-TrkA antibody
    
19. 10x Hanks’ balanced salt solution (HBSS) w/o PR, sod.bicarb, CaMg (Mediatech, catalog number: 20-023-CV )
    
20. Penicillin-streptomycin (100x) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
    
21. Neurobasal-A medium (Thermo Fisher Scientific, Gibco^TM, catalog number: 10888022 )
    
22. Fetal bovine serum (FBS) (Qmega Scientific, catalog number: FB-02 or various suppliers) (heat in activation before use)
    
23. 50x B27 supplement (Thermo Fisher Scientific, Gibco^TM, catalog number: 17504044 )
    
24. 100x GlutaMAX (Thermo Fisher Scientific, Gibco^TM, catalog number: 35050061 )
    
25. Mouse purified NGF as previously published (Mobley et al., 1986)
    
26. Dissection buffer (see Recipes)
    
27. Plating media (see Recipes)
    
28. Maintenance media (see Recipes)
    

Equipment

1. Sterile cell culture hood (Esco Micro, model: Class II Type A2 )
   
2. Scissors
   
3. #5 biologie forces and small spring scissors (Fine Science Tools)
   
4. Pipette
   
5. Water bath (Thermo Fisher Scientific, Thermo Scientific^TM, model: Precision^TM Model 281 )
   Note: This product has been discontinued.
   
6. Cell culture incubator (Panasonic, model: MCO-19AIC UV-PA )
   
7. Dissecting microscope (Leica, model: Leica S8 AP0 )
   
8. Cell culture centrifuge (Thermo Fisher Scientific, Thermo Scientific^TM, model: CL2 )
   
9. Autoclave (STERIS, model: AMSCO^® Evolution^® Steam Sterilizer )
   

Procedure