Materials and Reagents

1. Mouse mammary tumor
   
2. DMEM (Life Technologies, Gibco^®)
   
3. 2.5 mg/ml trypsin (0.06 g/25 ml) (Sigma-Aldrich, catalog number: T4799-10G )
   
4. 5 mg/ml albumin (125 mg/25 ml) (Thermo Fisher Scientific, catalog number: BP1600-100 )
   
5. 850 units/ml of collagenase type II (0.064 g/25 ml) (Worthington Biochemical, catalog number: 46J8959-A or 4176)
   
6. EGF (Pepro Tech, Catalog number: 100-15 )
   
7. Penicillin/Streptomycin (Pen/Strep) (Life Technologies, Gibco^®)
   
8. F12 (Life Technologies, Gibco^®)
   
9. Gentamycin (50 μg/ml)
   
10. Fetal Bovine Serum (Life Technologies, Gibco^®)
    
11. Insulin (Sigma-Aldrich, catalog number: I1882 )
    
12. Hydrocortisone (Sigma-Aldrich, catalog number: H0135 )
    
13. Gentamycin (Sigma-Aldrich, catalog number: G1397 )
    
14. Digestion buffer (Collagenase solution) (see Recipes)
    
15. Wash buffer (see Recipes)
    
16. Growth Media (see Recipes)
    

Equipment

1. 100 mm or 60 mm cell culture plate
   
2. Sterile single edged razor blade
   
3. Sterile scissors and tweezers
   
4. 10 ml beaker/flask with a sterile small stirrer bar
   
5. Cell strainers (BD, Falcon^TM Cell Strainers for 50 ml Conical Tubes, 100 μm)
   
6. Centrifuge 250 x g
   
7. 15 ml centrifuge tube
   
8. Tissue culture freezer
   
9. 37 °C with 5% CO₂ incubator
   

Procedure