TITLE: Gut prep of intestinal immune cells
AUTHORS: Connor Monahan

[DESCRIPTION]
This protocol details the procedure of purification of immune cells from the mouse small intestine.

[STEPS]
SECTION: Procedure
1. 
 
Cut out the entire small intestine – from stomach to caecum.
 

SECTION: Procedure
2. Place in ice cold PBS, move on to the next mouse.
SECTION: Procedure
3. Continue removing all fat and connective tissue.
SECTION: Procedure
4. Cut out the Peyer’s patches.
 

SECTION: Procedure
5. Cut open the intestine longitudinally.
SECTION: Procedure
6. Place on a wet paper towel, use rounded forceps to scrape along the mucosa, removing mucous, bacteria, etc.
SECTION: Procedure
7. Place in a 50 mL conical tube with PBS, invert tube a few times.
SECTION: Procedure
8. Pour of the supernatant, refill with fresh PBS.
SECTION: Procedure
9. Repeat the PBS washes 5-6 more times until there is no visible debris.
SECTION: Procedure
10. Cut the intestine in large fragments.
SECTION: Procedure
11. Place the fragments in a 15 mL conical tube with 5-10 mL cell dissociation solution.
SECTION: Procedure
11.1. To make 120 mL of cell dissociation solution (adjust as necessary):  
      HBSS  114.5 mL    0.5 M EDTA  1.2 mL    1M HEPES  1.2 mL    FBS  3.12 mL  

SECTION: Procedure
12. Incubate for   on the rotator.

SECTION: Procedure
13. Vortex well for 25 s, take out the supernatant with a metal strainer, keep the pieces, discard the supernatant.

SECTION: Procedure
14. Repeat steps 10-12.
SECTION: Procedure
15. Collect the fragments, rinse in HBSS in a small petri dish, cut using a razor blade (~1 mm2).
SECTION: Procedure
16. Digest for 20 min at 37 °C with slow rotation in 5 mL digestion mix.

SECTION: Procedure
16.1. To make 180 mL of digestion mix (adjust as necessary): 
      RPMI  126.6 mL    FBS  9 mL    DNase I  720 ul    Dispase (5U/mL)  18 mL    Collagenase D (Roche)  360 ul  
From 500 mg/mL stock solution, to make working conc of 1 mg/mL. 
SECTION: Procedure
17. Vortex well for 30 s.
SECTION: Procedure
18. Collect the supernatant by filtering through   strainer in 50 mL falcon tube at Room temperature to avoid cold temperature shock. Keep it on ice afterwards.
SECTION: Procedure
19. Put remaining tissue fragment back into the same tube with 5 mL of new digestion mix.
SECTION: Procedure
20. Repeat steps 16-18 two more times.
SECTION: Procedure
21. Combine all appropriate supernatants.
SECTION: Procedure
22. Centrifuge at 3000 rpm, 10 min, 4 °C.

SECTION: Procedure
23. Prepare Percoll (adjust amounts as necessary).
    a. 100% Percoll = 45 mL stock Percoll + 5 mL 10X PBS (50 mL total).
    b. 80% Percoll = 24 mL 100% Percoll + 6 mL 10% RPMI-FBS (30 mL total).
    c. 40% Percoll = 20 mL 100% Percoll + 30 mL 10% RPMI-FBS (50 mL total).
SECTION: Procedure
24. Add 5 mL of 80% Percoll to the bottom of a 15 mL conical tube.
SECTION: Procedure
25. Resuspend with LPL cells in 1 mL of 40% Percoll to get homogenous solution, then add the remaining 9 mL.
SECTION: Procedure
26. Gently add 10 mL of the 40% Percoll cell solution on top of the 80% Percoll in the tube.
SECTION: Procedure
27. Centrifuge at 2500 rpm, 20 min, without brake (accel 1, decel 0).

SECTION: Procedure
28. Collect and discard the top layer of epithelium and debris.
SECTION: Procedure
29. Collect white cell layer at the interface between the 40% and 80% Percoll.
SECTION: Procedure
30. Dilute with 10% RPMI-FBS, invert a few times to mix well.
SECTION: Procedure
31. Pellet by centrifugation for 2000 rpm, 7 min, 4 °C.

SECTION: Procedure
32. Aspirate SN and resuspend cells FACS buffer, then start FACS stain.