Materials and Reagents

1. Mice 13-14 days pregnant
   
2. Phosphate buffered saline (PBS), without Ca²⁺ and Mg²⁺ (Life Technologies, Gibco^®, catalog number: 10010 )
   
3. Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4.5 g/L D-glucose (Life Technologies, Gibco^®, catalog number: 11960044 or Mediatech, catalog number: 15-017-CV )
   
4. Fetal bovine serum (Atlanta Biologicals, catalog number: S11550 )
   
5. L-glutamine (Life Technologies, Gibco^®, catalog number: 25030-149 )
   
6. 1x penicillin-streptomycin solution (Gibco, catalog number: 15140-148 )
   
7. 0.25% trypsin-EDTA (Life Technologies, Gibco^®, catalog number: 25200 )
   
8. 70% ethanol
   Note: Prepare in a sterile container with sterile distilled water.
   
9. Dimethyl Sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
   
10. MEF culture medium with 10% fetal bovine serum (see Recipes)
    

Equipment

1. Plastic dissecting board or thick pad of blotting paper (such as Schleicher and Schuell GB004 paper)
   
2. Masking tape or push pins
   
3. Sterile Fine scissors (at least 3)
   
4. Sterile Fine forceps (at least 3)
   
5. Sterile 4 x 4 gauze pads (available from vendors of medical supplies)
   
6. Sterile scalpel blades or single-edge razor blades
   
7. Examination gloves
   
8. Petri dishes (100 mm) (BD Biosciences, Falcon^®, catalog number: 351029 , or equivalent)
   
9. Sterile plastic serological pipettes, 5 and 10 ml
   
10. Sterile disposable tubes (50 ml) (BD Biosciences, Falcon^®, catalog number: 352070 , or equivalent)
    
11. T75 tissue culture flasks (Corning, catalog number: 430641 , or equivalent)
    
12. 37 °C 5% CO₂ tissue culture incubator
    
13. Tissue culture hood
    
14. Inverted microscope
    
15. Cryovials (such as Nunc^®, catalog number: 375418 )
    

Procedure