Materials and Reagents

1. 12-well MatTek plate with 1.5 coverslip thickness (MATTEK, catalog number: P12G-1.5-14-F )
   
2. 18 G non-flexible stainless steel oral gavage needle (Cadence, Inc., catalog number: 7906 )
   
3. 10 ml syringe (Thermo Fisher Scientific, Fisher Scientific^TM, catalog number: 14-823-2A )
   
4. 50 ml sterile Falcon tubes (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 339653 )
   
5. Sterile 12-well cell culture dish (Sigma-Aldrich, catalog number: CLS3513 )
   
6. 15 ml sterile Falcon tubes (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 339651 )
   
7. Pre-chilled sterile pipette tips
   Notes:
   1. Any tips that are sterile and can be used with the lab’s pipettors.
   2. These tips can be placed in a -20 °C freezer for 30 min or in a 4 °C refrigerator one hour prior to plating.
8. 70 μM cell strainer (Thermo Fisher Scientific, Fisher Scientific^TM, catalog number: 08-771-2 )
   
9. C57BL/6 mouse (male or female, 6-8 weeks of age)
   
10. Isofluorane (Allivet, catalog number: 50562 )
    
11. Ice cold sterile 1x DPBS without calcium or magnesium (Thermo Fisher Scientific, Gibco^TM, catalog number: 14190-235 )
    
12. UltraPure EDTA (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 15576-028 )
    
13. Sucrose (Sigma-Aldrich, catalog number: S0389 )
    
14. D-Sorbitol (Sigma-Aldrich, catalog number: S1876 )
    
15. Matrigel^® basement membrane matrix (Corning, catalog number: 356237 )
    Note: Thaw at 4 °C the night before use.
    
16. Epidermal growth factor (EGF) (R&D Systems, catalog number: 2028-EG-200 )
    
17. Noggin (R&D Systems, catalog number: 1967-NG-025/CF )
    
18. R-spondin (R&D Systems, catalog number: 3474-RS-050 )
    
19. Wnt3A (R&D Systems, catalog number: 1324-WN-010 )
    
20. Advanced DMEM/F12 (Thermo Fisher Scientific, Gibco^TM, catalog number: 12634-010 )
    
21. L-glutamine (Thermo Fisher Scientific, Gibco^TM, catalog number: 25030-081 )
    
22. Penicillin-streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140-148 )
    
23. 1 M HEPES (pH 7.0-7.6, Sterile filtered) (Sigma-Aldrich, catalog number: H0887 )
    
24. N2 supplement (R&D Systems, catalog number: AR003 )
    
25. B27 supplement (Thermo Fisher Scientific, Gibco^TM, catalog number: 17504-044 )
    
26. Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10437-028 )
    
27. Click-iT EdU cell proliferation assay (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: C10337 )
    
28. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
    
29. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A8531 )
    
30. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
    
31. TO-PRO-3 Iodide (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: T3605 )
    
32. Chelation buffer, prepare fresh (see Recipes)
    
33. Shaking buffer (see Recipes)
    
34. Minigut culture media (see Recipes)
    
35. 2% Formaldehyde (see Recipes)

Equipment

1. Scissors for dissection (Fisher Scientific, catalog number: 08-951-20 )
   
2. Sorvall Legend X1R Centrifuge (or other large refrigerated centrifuge)
   
3. 37 °C, 5% CO₂ cell culture incubator (Thermo Fisher Scientific, model: HERAcell 150i )
   
4. LSM 510 META Inverted (or other inverted laser scanning) confocal microscope with 647 (TO-PRO-3) and 488 (EdU) emission filters
   

Software

1. ImageJ Image Analysis Software

Procedure