Materials and Reagents

1. Rat Tail Collagen I (Life Technologies, Invitrogen™, catalog number: A10483 )
   
2. Fetal Bovine Serum (FBS)
   
3. Pen-Strep
   
4. Hanks Balanced Salt Solution (HBSS) (Life Technologies, Gibco^®, catalog number: 14065 )
   
5. E.Z.N.Z Total RNA Kit I (Omega Bio-Tek, catalog number: R6834-01 )
   
6. BME-phenol blue
   
7. Xylene
   
8. 10x RPMI 1640 medium (Life Technologies, Gibco^®, catalog number: 31800-022 ) (see Recipes)
   
9. 4.2% NaHCO₃ (see Recipes)
   
10. Collagenase P (Roche, catalog number: 11213857001 ) (see Recipes)
    
11. 100x Soybean Trypsin Inhibitor (STI) (Sigma-Aldrich, catalog number: T6522 ) (see Recipes)
    
12. 2,000x Dexamethasone (Dex) (Sigma-Aldrich, catalog number: D4902 ) (see Recipes)
    
13. 3D-culture medium (see Recipes)
    

Equipment

1. Centrifuge (Beckman Coulter, model: GS-15R or equivalent, rotor S4180)
   
2. Standard 5% CO₂ tissue culture incubator
   
3. 24-well tissue culture plates
   
4. 37 °C shaker
   
5. 6 cm culture dish
   
6. Dissection scissors
   
7. 15 ml plastic screw cap tube
   
8. Laminar flow hood
   
9. Rocking platform
   
10. Tissue processor
    
11. Polypropylene Mesh (105 μm & 500 μm sizes) (Spectra/Mesh) (Fisher Scientific, catalog number: 146436 and 146418 )
    Note: Cut meshes into 10 x 10 cm squares and sterilize by autoclaving.
    
12. Homogenizer spin column (Omega Bio-Tek, catalog number: HCR003 )
    
13. Tissue-Teck Biopsy Uni-cassette (Sakura, catalog number: 4087)
    
14. Falcon tube (15 ml and 50 ml)
    
15. Nanodrop spectrophotometer
    

Procedure