Dissociated Retinal Cell Culture

Materials and Reagents

1. Animals
   We typically use retinas from albino Sprague Dawley rats. This method works for culturing rat retinas aged between embryonic day 17 to post-natal day 1. Younger and older retinal cells or retinal cells from mouse retinas do not seem to survive as well under these conditions, but can also be cultured.
   
2. DPBS (Life Technologies, catalog number: 14040 )
   
3. Neurobasal medium (Life Technologies, catalog number: 21103-049 )
   
4. DMEM/F12 medium (Life Technologies, catalog number: 10565-018 )
   
5. B27 supplement (Life Technologies, catalog number: 17504-044 )
   
6. Pen/Strep (P/S) (Life Technologies, catalog number: 15140-122 )
   
7. Poly L-lysine (PLL) (Sigma-Aldrich, catalog number: P-4707 )
   
8. Mouse Laminin (Life Technologies, catalog number: 23017-015 )
   
9. Papain (Worthington Biochemical, catalog number: LS003126 )
   
10. L-cystein crystal (Sigma-Aldrich, catalog number: C-7352 )
    
11. Trypsin Inhibitor (Roche Diagnostics, catalog number: 109878 )
    
12. 0.45 µm and 0.22 µm filters (Fisher Scientific, catalog number: SLHV033RS and SLGP033RS )
    
13. Hoechst 33342 (Life Technologies, Molecular Probes^®, catalog number : H3570 )
    
14. “Incomplete” RGM medium
    
15. Growth factors (see Recipes)
    1.  bFGF (Pepro Tech, catalog number: 100-18B )
       
    2. EGF (Pepro Tech, catalog number: 315-09 )
       
    3. NT-3 (Pepro Tech, catalog number: 450-03 )
       
    4. BDNF (Pepro Tech, catalog number: 450-02 )
16. 10x Lo Ovomucoid (see Recipes)
    
17. 20x 4% BSA (Sigma-Aldrich, catalog number: A-4161 ) (see Recipes)
    
18. 0.4% DNase (Worthington Biochemical, catalog number: LS002007 ) (see Recipes)
    
19. Insulin (2.5 mg/ml) (Sigma-Aldrich, catalog number: I-6634 ) (see Recipes)
    
20. cpt-cAMP (Sigma-Aldrich, catalog number: C-3912 ) (see Recipes)
    
21. N-acetyl cysteine (NAC) (Sigma-Aldrich, catalog number: A-9165 ) (see Recipes)
    
22. Forskolin (Sigma-Aldrich, catalog number: D-2438 ) (see Recipes)
    
23. N2 Supplement (see Recipes)
    
24. 4% Paraformaldehyde (Electron Microscopy Science, catalog number: 15710 ) (see Recipes)
    
25. Apo-Transferrin (Sigma-Aldrich, catalog number: T-1147 ) (see Recipes)
    
26. Progesterone (Sigma-Aldrich, catalog number: P-8783 ) (see Recipes)
    
27. Putrescine (Sigma-Aldrich, catalog number: P-5780 ) (see Recipes)
    
28. Sodium Selinite (Sigma-Aldrich, catalog number: S-5261 ) (see Recipes)
    

Equipment

1. 35 mm petri dish (BD Biosciences, Falcon^®, catalog number: 353001 )
   
2. 50 ml conical tube
   
3. CO₂ incubator
   
4. 15 ml conical tube
   
5. 100 mm petri dish
   
6. CO₂ chamber
   
7. Straight forcep
   
8. Fine forcep (Fine Science Tools, catalog number: 11252-23 )
   
9. Curved forcep (Fine Science Tools, catalog number: 91197-00 )
   
10. Sharp scissor
    
11. Laminar flow hood
    
12. Water bath
    
13. P1000 pipette
    
14. Microscope
    
15. Hemacytometer
    
16. Centrifuge
    

Procedure