Materials and Reagents

1. Mice
   
2. Collagenase Type IV (0.5 mg/ml) (Sigma-Aldrich, catalog number: C5138 ) or Liberase Blendzyme 2 (2 μg/ml) (Roche Diagnostics, catalog number: 1988433 )
   
3. RPMI 1640 (Life Technologies, catalog number: 11875-093 )
   
4. Gentamicin (10 μg/ml) (Life Technologies, catalog number: 15750-102 )
   
5. Type IV DNase I from bovine pancreas (25 units/ml) (Sigma-Aldrich, catalog number: D5025 ) or DNase I from bovine pancreas (25 units/ml) (Roche Diagnostics, catalog number: 11284932001 )
   
6. Fetal bovine serum heat inactivated (Life Technologies, catalog number: 10437-028 )
   
7. Red cells lysing buffer (see Recipes)
   
8. 1x phosphate buffered saline (PBS) (see Recipes)
   
9. 2x digestion medium (see Recipes)
   

Equipment

1. Syringes (BD Biosciences, catalog number: 301604 )
   
2. Centrifuge 5810 (Eppendorf)
   
3. 18-gauge needle (BD Biosciences, catalog number: 305180 )
   
4. Cell strainer (100-μm pore size) (Corning, catalog number: 352360 )
   
5. Sterile culture hood
   

Procedure