Materials and Reagents

1. Pregnant mouse (embryonic day: E18.5)
   
2. Borax (Sigma-Aldrich, catalog number: 71997 )
   
3. Boric acid (Sigma-Aldrich, catalog number: B6768 )
   
4. 50x B27 supplement (Life Technologies, Invitrogen^TM, catalog number: 17504-044 )
   
5. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
   
6. Dulbeccos modified Eagle medium (DMEM) (Life Technologies, Invitrogen^TM, catalog number: 31966-021)
   
7. Fetal bovine serum (FBS) (Life Technologies, Invitrogen^TM, catalog number: 16000-044 )
   
8. 100x glutamax (Life Technologies, Invitrogen^TM, catalog number: 35050-038)
   
9. 10x HBSS (Life Technologies, Invitrogen^TM, catalog number: 14185-052 )
   
10. 1 M MnCl₂ (Sigma-Aldrich, catalog number: M1787 )
    
11. Neurobasal medium (Life Technologies, Invitrogen^TM, catalog number: 21103-049 )
    
12. Penicillin-streptomycin (PS) (10,000 U/ml) (Life Technologies, Invitrogen^TM, catalog number: 15140-122 )
    
13. Dulbecco’s phosphate buffer saline (DPBS) with Ca²⁺ and Mg²⁺ (Sigma-Aldrich, catalog number: D8662 )
    
14. Poly-L-Lysine hydrobromide (Sigma-Aldrich, catalog number: P2636 )
    
15. Sterile water
    
16. Thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich, catalog number: M2128 )
    
17. 10x trypsine (Life Technologies, Invitrogen^TM, catalog number: 15090-046 )
    
18. Borate buffer (see Recipes)
    
19. 1 mg/ml poly-L-lysine (see Recipes)
    
20. First culture medium (see Recipes)
    
21. 1x HBSS (see Recipes)
    
22. Second culture medium (see Recipes)
    
23. MnCl₂ (0, 20, 50, 100 and 150 µM) (see Recipes)
    
24. 0.5 mg/ml MTT (see Recipes)
    

Equipment

1. One sterile curved plier and one sterile sharp plier (Figure 1)
   
2. One bag of sterile plastic Petri dishes (100 mm diameter)
   
3. 24 wells plate (sterile)
   
4. 96 wells plate (non sterile)
   
5. Syringe filter
   
6. Pipette P1000 with a P1000 cone and a P200 cone (sterile)
   
7. Neubauer or Malassez cell counting chamber
   
8. Eppendorf tubes
   
9. Cell scraper
   
10. Tissue culture hood
    
11. Optic microscope inside the hood
    
12. Water bath at 37 °C
    
13. Incubator at 37 °C (humidified atmosphere with 5% CO₂)
    
14. Microplate reader (Pherastar plus) (BMG LABTECH)
    
    
    Figure 1. Pliers for the hippocampi dissection
    

Procedure