Materials and Reagents

1. Petri dishes (diameter 5.5 cm to 9 cm)
   
2. Falcon tubes 50 ml and 15 ml
   
3. Eppendorf tubes 2 ml (sterilized by autoclave)
   
4. Pasteur pipettes (1 mm diameter)
   
5. Brush (da Vinci, model: Nova synthetics series 1570, #3 )
   
6. 12-well and 24-well tissue culture plates (growth-enhance treated, gamma-sterilised, free of pyrogens, free of DNA/RNA, DNase/RNase)
   
7. Filter discs (Sartorius AG, catalog number: FT-3-205-055 )
   
8. Falcon cell strainers 40 µm and 100 µm (Thermo Fisher Scientific, catalog number: 08-771-1 and 08-771-19 )
   
9. Hanging insert for 24-well plates, 1 µm PET (Merck Millipore Corporation, catalog number: PIRP12R48 )
   
10. 27G ¾ needle (Braun, Sterican^TM, catalog number: 4657705 )
    
11. Mice at the age of six weeks
    Note: 6-8 mice for endothelial and pericyte isolation, at least 1 mouse for neurosphere co-culture assay
    
12. Mice at the age of four weeks
    Note: 4 mice for astrocyte isolation are needed for one preparation.
    
13. Distilled water
    
14. Poly-D-lysine hydrobromide (PDL) (Sigma-Aldrich, catalog number: P7280 )
    
15. Laminin (Sigma-Aldrich, Roche, catalog number: R11243217001 )
    
16. 70% ethanol
    
17. HBSS (Thermo Fisher Scientific, Gibco^TM, catalog number: 14175 )
    
18. Dextran from Leuconostoc mesenteroides average molecular weight 150,000 (Sigma-Aldrich, catalog number: D4876 )
    
19. DMEM/F-12 without glutamine (store at 4 °C) (Thermo Fisher Scientific, Gibco^TM, catalog number: 21331020 )
    
20. DMEM/F-12 with L-glutamine and 15 mM HEPES (store at 4 °C) (Thermo Fisher Scientific, Gibco^TM, catalog number: 11330032 )
    
21. B27 (Thermo Fisher Scientific, Gibco^TM, catalog number: 0080085SA )
    
22. MCDB131 Media (Thermo Fisher Scientific, catalog number: 10372-019 )
    
23. Glutamax (100x stock) (store at 4 °C) (Thermo Fisher Scientific, CTS^TM, catalog number: A12860-01 )
    
24. Insulin-transferrin-selenium (ITS) premix (store at -20 °C) (Thermo Fischer Scientific, Corning^TM, catalog number: CB-40351 )
    
25. Non-essential amino acids (100x stock) (store at 4 °C) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 11140-035 )
    
26. Pen/Strep 100,000 U/ml (-20 °C) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140 )
    
27. Heparin (store at 4 °C) (MP Biomedicals, catalog number: 0210193125 )
    
28. Collagenase A (Sigma-Aldrich, Roche, catalog number: 10103578001 )
    
29. Fetal bovine serum (FBS) (-20 °C) (Merck Millipore Corporation, Biochrom, catalog number: S0613 )
    
30. Plasma derived platelet poor serum (Sigma-Aldrich, catalog number: P2918 )
    
31. Human EGF (store aliquots at -20 °C) (PEPROTECH, catalog number: AF100-15 )
    
32. Human FGF-basic (store aliquots at -20 °C) (PEPROTECH, catalog number: 100-18B )
    
33. Human VEGF (store aliquots at -20 °C) (PEPROTECH, catalog number: 100-20 )
    
34. Accutase (store at 4 °C) (GENTAUR, PAA Laboratories GmbH, catalog number: L11-007 )
    
35. Neuronal tissue dissociation kit (P) (Miltenyl Biotec, catalog number. 130-092-628 )
    
36. Trypsin/EDTA (store at -20 °C) (Merck Millipore Corporation, Biochrom, catalog number: L2153 )
    
37. PBS with 2% FBS (see Recipes)
    
38. 30% dextran solution (see Recipes)
    
39. Endogrow media (see Recipes)
    
40. Astrocyte media (see Recipes)
    
41. Pericyte/smooth muscle cell media (see Recipes)
    
42. Neurosphere media (see Recipes)
    

Equipment

1. Dissection tools: scissors, small spatula, fine curved forceps and scalpel (see Figure 2B)
   
2. 7 ml Dounce tissue grinder (Capitol Scientific, Wheaton, catalog number: 357542 )
   
3. Autoclave
   
4. Vacuum pump
   
5. Dounce homogenizer
   
6. Dissection microscope (Olympus, model: SZ61 )
   
7. Inverted microscope (Olympus, model: CKX41 )
   
8. Scale plate insert into the eyepiece of the microscope (Olympus)
   
9. Cooling centrifuge fitting 2 ml tubes (Eppendorf, model: 5430R )
   
10. Swing bucket centrifuge for 15 ml tubes (Eppendorf, model: 5810R )
    
11. Incubator at 37 °C with 5% CO₂
    
12. Tube rotator or orbital shaker
    

Procedure