Materials and Reagents

1. Mouse-adapted prion infected mice brains (terminally ill CD-1 female mice intracerebrally inoculated with mouse adapted scrapie, at ca. 6 months of age)
   
2. Ethanol (99.5% EtOH) (Nacalai Tesque, catalog number: 14713-95 )
   
3. Dulbecco's phosphate buffered saline without Ca and Mg (D-PBS) (Nacalai Tesque, catalog number: 14249-95 )
   
4. Hank's balanced salt solution (HBSS) (Sigma-Aldrich, catalog number: H8264 )
   
5. Dulbecco's modified Eagle's medium/nutrient F-12 Ham (DMEM/F12 Ham) (Sigma-Aldrich, catalog number: D8437 )
   
6. N-2 supplement (Life Technologies, catalog number: 17502-048 )
   
7. Bovine serum albumin solution (30% BSA in D-PBS) (Sigma-Aldrich, catalog number: A9576 )
   
8. Accutase (Innovative Cell Technologies, catalog number: AT-104 )
   
9. 99% Acetone (Nacalai Tesque, catalog number: 00310-95 )
   
10. Water for embryo transfer (sterile-filtered) (Sigma-Aldrich, catalog number: W1503 )
    
11. 35% hydrochlolic acid (HCl) ( Nacalai Tesque, catalog number: 18321-05 )
    
12. Penicillin-Streptomycin (Sigma-Aldrich, catalog number: P0781 )
    
13. Epidermal growth factor human (EGF) (Sigma-Aldrich, catalog number: E9644 ) (see Recipes)
    
14. Fibroblast growth factor-basic human (bFGF) (Sigma-Aldrich, catalog number: F0291 ) (see Recipes)
    
15. Neurosphere basal medium (see Recipes)
    
16. Fibronectin (Sigma-Aldrich, catalog number: F4759 ) (see Recipes)
    
17. Glass wash solution (see Recipes)
    
18. Differentiation-inducing medium (see Recipes)
    

Equipment

1. Falcon 15 ml conical centrifuge tubes (Corning, catalog number: 352096 )
   
2. Falcon 50 ml conical centrifuge tubes (Corning, catalog number: 352070 )
   
3. Corning primaria 6 well clear multiwell plate (Corning, catalog number: 353846 )
   
4. Nunc 60mm dish with HydroCell surface (Thermo Fisher Scientific, catalog number: 174912 )
   
5. Glass coverslips (22 x 22 mm) (Asahi Techno Glass Corporation, catalog number: 2918 )
   
6. ART 1000 REACH tips (Thermo Fisher Scientific, catalog number: 2079 )
   
7. Multi-beads shocker cell disruptor (Yasui Kikai Corporation)
   
8. 2 ml screw-cap micro centrifuge tubes-freestanding (Sarstedt K.K., catalog number: 72.664.002 )
   
9. Zirconia ball (1 mm diameter) (As One YTZ1)
   
10. Plastic square dishes (Eiken Chemical, model: AW2000 )
    
11. 1.5 ml screw-cap micro centrifuge tubes (Sarstedt K.K., catalog number: 72.687 .028J)
    
12. Sealed sonicator (Biorupter^®, model: UCD250 )
    
13. Centrifuge (Kubota Corporation, model: 5220 )
    
14. CO₂ incubator (set at 37 °C and 5% CO₂-95% air)
    
15. Reciprocal shaker (Taitec, model: NR-1 )
    
16. Bio-safety cabinet (ORIENTAL GIKEN, model: LAD-1300XB3 )
    
17. Deep freezer (set at -80 °C)
    
18. Fine forceps (NAPOX A-5, 110 mm; NAPOX MA-45, 110 mm)
    
19. C-Chip disposable hemocytometer (iN CYTO, DHC-N01 Neubauer Improved)
    
20. Electric balance (Mettler Toledo, model: AB104-S )
    

Procedure