Materials and Reagents

1. EasySep^® "Do-It-Yourself" Selection Kit (STEMCELL Technologies, catalog number: 18098 )
   
2. Mouse IgG1 P75^NTR antibody (Abcam, catalog number: ab8877 )
   
3. 2% Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F4135 ) in PBS (2 ml in 98 ml  of PBS,10 mM)
   
4. Phosphate buffered saline (PBS) (Life Technologies, catalog number: 00-3002 )
   
5. Dissolve tablets in 100 ml of distilled H₂O , The buffer contains 10 mM phosphate, 150 mM sodium chloride (pH 7.3-7.5 )
   
6. 10x Trypsin solution (Life Technologies, Invitrogen^TM , catalog number: 15090046 )
   
7. Low-glucose DMEM (Life Technologies, Invitrogen^TM , catalog number: 21885025 )
   
8. 5% v/v FBS (Sigma-Aldrich, catalog number: F4135)
   
9. 2 mM L-glutamine (Life Technologies , Invitrogen^TM, catalog number: 25030024 )
   
10. Bovine serum albumin Pathocyte (MP Biomedicals, catalog number: 810111 )
    
11. Bovine pancreatic insulin (Sigma-Aldrich, catalog number: I-5500 )
    
12. Human transferrin (Sigma-Aldrich, catalog number: T-2252 )
    
13. Progesterone (Sigma-Aldrich, catalog number: P-0130 )
    
14. Putrescine (Sigma-Aldrich, catalog number: P7505 )
    
15. l-thyroxine (Sigma-Aldrich, catalog number: T-2501 )
    
16. Selenium (Sigma-Aldrich, catalog number: S-1382 )
    
17. 3,3’,5-triiodo-l-thyronine (Sigma-Aldrich, catalog number: T-2752 )
    
18. FGF2 (25 ng/ml ) (Peprotech , catalog number: 100-18B )
    
19. Heregulin β-1 (50 ng/ml ) (R&D Systems, catalog number: 396-HB-050 )
    
20. Forskolin (5 x 10^-7 M) (Sigma-Aldrich , catalog number: F6886 )
    
21. Gentamicin solution (50 mg/ml use at 1 ml/l) (Sigma-Aldrich , catalog number: G1397 )
    
22. L15 media (Leibovitz medium) (Sigma-Aldrich, catalog number: L1518 )
    
23. 1.33% collagenase (MP Biomedicals UK, catalog number: 195109 Type I )
    
24. Plastic bijou bottle (Sterilin ) (Thermo Fisher Scientific , catalog number: 129A )
    
25. Bovine pancreas DNAse (Sigma-Aldrich , catalog number: D4263 )
    
26. Bovine serum albumin fraction v (Sigma-Aldrich , catalog number: A2153 ) 
27. Astrocyte-conditioned media (ACM; use at 1:5) ACM is fresh serum-free media (DMEM-BS) collected from a confluent astrocyte monolayer for 48 h (noble  and Murray, 1984; Alexander et al., 2002 )
    
28. Poly-L-Lysine (Sigma-Aldrich, catalog number: P1274 )
    
29. 70% ethanol
    
30. 10% v/v serum free DMEM-Bottenstein and Sato (DMEM-BS) (Bottenstein et al., 1979) (see Recipes)
    

Equipment

1. EasySep^® Magnet Catalog (STEMCELL technologies, catalog number: 18000 )
   
2. FACS tubes 5 ml Polystyrene Round-Bottom Tubes (BD Biosciences, catalog number: 352058 )
   
3. 50 ml centrifuge tubes (BD Biosciences, catalog number: 352070 )
   
4. 40 μm cell strainers (BD Biosciences, catalog number: 352340 )
   
5. 1.5 ml polypropylene microcentrifuge tube (Griener Bio-One GmbH, catalog number: 616201 )
6. Poly-l-lysine coated (13.3 μg/ml, MW-100,000) (Sigma-Aldrich, catalog number: P1274), T25 cm² tissue culture flasks (Greiner Bio-One GmbH, catalog number: 690175 )
   
7. Nunc sterile centrifuge tube (Thermo Fisher Scientific, catalog number: 339651 )
   
8. 25 cm² flask (Greiner Bio-One GmbH, catalog number: 690160 )
   
9. 40 μm cell strainer (BD Biosciences, Falcon^®, catalog number: 352340)
   
10. 37 °C incubator
    
11. Plastic bijou bottle (Thermo Fisher Scientific, Sterilin^®, catalog number: 129A)
    
12. 21 gauge needle (BD Biosciences , catalog number: 305167 ) 
    
13. 23 G needle (BD Biosciences , catalog number: 305143 ) 
    

Procedure