Materials and Reagents

1. Newborn mice (age at one day after birth)
   Present culture method can be applied to any mouse strains, regardless of sex. We used prion protein overexpressing transgenic mouse and prion protein-deficient mouse for our present study (Iwamaru et al., 2013).
   
2. Dulbecco's phosphate buffered saline without Ca and Mg (D-PBS) (Nacalai Tesque, catalog number: 14249-95 )
   
3. Di‐sodium dihydrogen ethylenediamine tetraacetate dihydrate (EDTA) (Nacalai Tesque, catalog number: 151-11 )
   
4. Hank's balanced salt solution (HBSS) (Sigma-Aldrich, catalog number: H8264 )
   
5. Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham (DMEM/F12 Ham) (Sigma-Aldrich, catalog number: D8437 )
   
6. N-2 supplement (Life Technologies, catalog number: 17502-048 )
   
7. Penicillin-Streptomycin (Sigma-Aldrich, catalog number: P0781 )
   
8. Fetal bovine serum (FBS) (Hyclone, catalog number: SH30070.03 )
   
9. Accutase (Innovative Cell Technologies, catalog number: AT104 )
   
10. Trypsin (Sigma-Aldrich, catalog number: T8003 ) (see Recipes)
    
11. Deoxyribonuclease I (DNase I) (Worthington Biochemical Corporation, catalog number: LS002139 ) (see Recipes)
    
12. Epidermal growth factor human (EGF) (Sigma-Aldrich, catalog number: E9644 ) (see Recipes)
    
13. Fibroblast growth factor-basic human (bFGF) (Sigma-Aldrich, catalog number: F0291 ) (see Recipes)
    
14. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9576 ) (see Recipes)
    
15. Neurosphere basal medium (see Recipes)
    

Equipment

1. Falcon 15 ml conical centrifuge tube (Corning, catalog number: 352096 )
   
2. Falcon 100 mm cell culture dish (Corning, catalog number: 353003 )
   
3. Nunc 60 mm dish with HydroCell surface (Thermo Fisher Scientific, catalog number: 174912 )
   
4. Disposable micro homogenizer (TaKaRa Bio, TaKaRa BioMasher Standard, catalog number: 9791A )
   
5. Aerosol resistant tip (ART 1000 REACH tips) (Thermo Fisher Scientific, model: 2079 )
   
6. Membrane filter (EMD Millipore, Millex-GV 0.22 μm, 33 mm)
   
7. Dissecting microscope (Olympus, model: SZH10 )
   
8. Centrifuge (KUBOTA, model: 5220 )
   
9. CO₂ incubator (set at 37 °C and 5% CO₂-95% air)
   
10. Reciprocal shaker (TAITEC, model: NR-1 )
    
11. Laminar flow hood (W800 x H1050 x D500 mm) (Panasonic Corporation, SANYO, model: MCV-710ATS )
    
12. Scissors (NAPOX B-5H, 154 mm; NAPOX B-1112H, 110 mm), fine forceps (NAPOX A-5, 110 mm; NAPOX MA-45, 110 mm), spatula (Laboran 9-891-03, 180 mm)
    
13. Ice bucket
    

Procedure