Materials and Reagents

1. 15 ml Falcon tube (Corning, Falcon^®, catalog number: 352095 )
   
2. Petri dish (Thermo Fisher Scientific, Fisher Scientific, catalog number: FB0875713 )
   
3. Nunclon^TM tissue culture dish (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 172931 )
   
4. New born mice (postnatal day 0)
   
5. 70% sterile ethanol (prepared from ethanol 190 proof) (Decon Labs, catalog number: 2801 )
   
6. Distilled water (Thermo Fisher Scientific, Gibco^TM, catalog number: 15230162 )
   
7. PBS without Ca²⁺ Mg²⁺ (Thermo Fisher Scientific, Gibco^TM, catalog number: 20012050 )
   
8. Trypsin-EDTA (Thermo Fisher Scientific, Gibco^TM, catalog number: 25200072 )
   
9. DMEM (Thermo Fisher Scientific, Gibco^TM, catalog number: 11995-065 )
   
10. Fetal bovine serum (GE Healthcare, Hyclone^TM, catalog number: SH30396.03 )
    
11. Penicillin-streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
    
12. DMSO (Sigma-Aldrich, catalog number: D2650 )
    
13. Liquid nitrogen
    
14. Dispase II (Roche Diagnostics, catalog number: 04942078001 )
    
15. Sorbitol
    
16. Collagenase (Type L) (Sigma-Aldrich, catalog number: C8170 ), sterile-filtered, dissolved in PBS and stored at -20 °C. 

    
17. Hyaluronidase
    
18. Disapse solution (see Recipes)
    
19. Digestion buffer (see Recipes)
    

Equipment

1. CO₂ cabinet
   
2. Chemical hood
   
3. Tissue culture hood
   
4. Phase-contrast inverted microscope (Electron Microscopy Sciences)
   
5. Spring scissor (Fine Science Tools, catalog number: 15009-08 )
   
6. Forceps (Fine Science Tools, catalog number: 00108-11 )
   
7. Benchtop centrifuge (Hettich Lab Technology, model: Rotina 380 )
   
8. Pipettes
   
9. Hemocytometer (Hausser Scientific)

   
10. CO₂ incubator
    

Procedure