Generation of Mouse Thyroid Calcitonin-producing Cell Tumors from Primary Mouse Tumors

Materials and Reagents

1. 12 well cell culture plate (Thermo Fisher Scientific, catalog number: 150628 )
   
2. 100 mm cell culture dish (Thermo Fisher Scientific, catalog number: 172931 )
   
3. 5 ml centrifuge tube (Thermo Fisher Scientific, catalog number: 339650 )
   
4. Phosphate buffered saline (PBS) (pH 7.2) (Life Technologies, catalog number: 20012027 )
   Note: Currently, it is “Thermo Fisher Scientific, Gibco^TM, catalog number: 20012027”.
   
5. 100x Antibiotic-Antimycotic (Life Technologies, catalog number: 15240062 )
   Note: Currently, it is “Thermo Fisher Scientific, Gibco^TM, catalog number: 15240062”.
   
6. Dulbecco’s modified Eagle's medium (DMEM) (Wako Pure Chemical Industries, catalog number: 04330085 )
   
7. 3,000 U/ml Collagenase from Clostridium histolyticum (Sigma-Aldrich, catalog number: C5138 )
   
8. 1,000 U/ml Hyaluronidase (Wako Pure Chemical Industries, catalog number: 08006201 )
   
9. 20 mg/ml Deoxyribonuclease I from bovine pancreas (Sigma-Aldrich, catalog number: DN25 )
   
10. Fetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: SH3091003 )
    Note: Currently, it is “GE Healthcare, catalog number: SH3091003”.
    
11. Penicillin-Streptomycin Mixed Solution (Nakarai tesque, catalog number: 2625384 )
    
12. 0.5 g/l-Trypsin/0.53 mmol/l-EDTA Solution, with Phenol Red (Nakarai tesque, catalog number: 3277834 )
    
13. 7-AAD (BD Pharmingen, catalog number: 5168981E )
    
14. CELLBANKER1 (Nippon Zenyaku Kogyo Co., ZENOAQ, catalog number: CB011 )
    
15. Wash buffer (see Recipes)
    
16. Digestion solution (see Recipes)
    
17. Growth medium (see Recipes)
    

Equipment

1. Surgical scissors and forceps
   
2. Pipettes
   
3. Centrifuge
   
4. Cell culture hood
   
5. 37 °C, 5% CO₂ cell culture incubator
   
6. Microscope
   
7. FACS Aria ll flow cytometer (BD Biosciences)
   

Procedure