Materials and Reagents

1. Petri dishes (100 mm)
   Note: Any Petri dish can be used, according to each researcher’s preference and availability.
   
2. Falcon tube
   
3. Transfer pipettes 3.5 ml (SARSTEDT, catalog number: 86.1172.001 )
   
4. 50 ml conical tubes (SARSTEDT, catalog number: 62.547.004 )
   
5. Syringe filters, 0.2 μm pore size (SARSTEDT, catalog number: 83.1826.001 )
   
6. Cell strainers 70 μm (Corning, Falcon^®, catalog number: 352350 )
   
7. Cell culture treated flasks (25 cm²) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 136196 )
   
8. Cell culture treated flasks (175 cm²) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 178883 )
   
9. Cell culture treated flasks (80 cm²) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 178905 )
   
10. Mice
    
11. Ethanol
    Note: Any ethanol can be used, according to each researcher’s preference and availability.
    
12. HBSS (Thermo Fisher Scientific, Gibco^TM, catalog number: 14170088 )
    
13. Antibiotic-antimycotic (100x) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15240096 )
    
14. EDTA (Thermo Fisher Scientific, Fisher Scientific, catalog number: 327205000 )
    
15. DTT (DL-Dithiothreitol) (Sigma-Aldrich, catalog number: D9779 )
    
16. DMEM (Thermo Fisher Scientific, Gibco^TM, catalog number: 11960044 )
    
17. Fetal bovine serum (FBS) (Biochrom, catalog number: S0115 )
    
18. Penicillin-streptomycin 100x (10,000 U/ml) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
    
19. L-glutamine (200 mM) (Thermo Fisher Scientific, Gibco^TM, catalog number: 25030-081 )
    
20. MEM non-essential amino acids solution (100x) (Thermo Fisher Scientific, Gibco^TM, catalog number: 11140-068 )
    
21. Phosphate-buffered saline (PBS) Dulbecco (10x) (Biochrom, catalog number: L1835 )
    
22. Amphotericin B (Sigma-Aldrich, catalog number: A2942 )
    
23. Collagenase from Clostridium histolyticum, type XI (Sigma-Aldrich, catalog number: C7657 )
    
24. Dispase II, neutral protease grade II, from Bacillus polymyxa (Roche Diagnostics, catalog number: 04942078001 )
    
25. Trypsin-EDTA (0.05%) (Thermo Fisher Scientific, Gibco^TM, catalog number: 25300054 )
    
26. FACS staining antibodies and material
    
    1. Vimentin-A647 (Abcam, catalog number: ab194719 )
       
    2. CD45-PE-Cy5 (BD, catalog number: 553082 )
       
    3. Intracellular fixation & permeabilization buffer set (eBioscience, catalog number: 88-8824-00 )
       
27. HBSS/antibiotics (see Recipes)
    
28. HBSS/EDTA/DTT (see Recipes)
    
29. Digestion solution (see Recipes)
    
30. DMEM medium (see Recipes)
    

Equipment

1. Dissection kit
   1. Two scissors (at least one medium size and one small)
      
   2. Two forceps
      Note: Any scissors and forceps can be used, according to each researcher’s preference and availability.
      
   3. A 10 ml syringe equipped with a 21-gauge needle for flushing the intestine (BD, Plastipak^TM, catalog number: 308063 )
      
2. Cell culture hood
   
3. Cell culture incubator, 37 °C, 5% CO₂
   
4. Shaking water-bath
   
5. Centrifuge compatible with 50 ml conical tubes
   
6. Bright-field inverted microscope for cell culture
   
7. Multicolor FACS analyser (BD, model: BD FACS Canto II )
   

Software

1. FlowJo software (Tree Star Inc.)

Procedure