Materials and Reagents

1. Mouse (e.g. Balb/c) 8-20 weeks old
   
2. Sterile PBS without Ca/Mg (Euroclone, catalog number: ECB4004L )
   
3. Pen/Strep: 100x solution of penicillin/streptomycin/fungizone (Life Technologies, Gibco^®, catalog number: 15240-062 )
   
4. Gentamycin (50 mg/ml solution) (Life Technologies, Gibco^®, catalog number: 15750-037 )
   
5. RPMI 1640 (with glutamine) (Lonza, catalog number: BE12-702F )
   
6. Fetal bovine serum (FBS) (heat inactivated) (Lonza, catalog number: DE14-801F )
   
7. 0.4% trypan blue (Sigma-Aldrich, catalog number: T8154 )
   
8. Phorbol 12-Myristate 13-Acetate (PMA) (Sigma-Aldrich, catalog number: P8139 - follow the manufacturer's instructions to prepare stock solutions)
   
9. Ionomycin (Sigma-Aldrich, catalog number: I9657 - follow the manufacturer's instructions to prepare stock solutions)
   
10. Common disinfectant (i.e. 70-90% ethanol, tincture of iodine)
    
11. PBS + 2% Pen/Strep (see Recipes)
    
12. RPMI + 2% Pen/Strep (see Recipes)
    
13. Complete medium (see Recipes)
    

Equipment

1. Soft wood tablet and pins
   
2. Aluminium foils (or another smooth, non-absorbent surface)
   
3. Three sterile small thin surgical scissors
   Note: In particular one for cutting the skin, the second one for cutting the muscular wall and the intestine, and the last one for removing PPs.
   
4. Four sterile small thin surgical tweezers, two straight and two curved (straight ones for cutting the skin, the muscular wall and the intestine; curved ones to expel faeces and to remove PPs)
   
5. 70 µm cell strainer (BD Biosciences, Falcon^®, catalog number: 352350 )
   
6. 2.5 ml syringes
   
7. 15 and 50 ml centrifuge tubes
   
8. 24-well cell culture plates
   
9. Refrigerated centrifuge
   
10. Sterile flow hood
    
11. Thermostatic water bath
    
12. Inverted microscope for cell cultures
    
13. 37 °C, 5% CO₂ cell culture incubator
    

Procedure