Gut prep of intestinal immune cells

Cut out the entire small intestine – from stomach to caecum.

Place in ice cold PBS, move on to the next mouse.

Continue removing all fat and connective tissue.

Cut out the Peyer’s patches.

Cut open the intestine longitudinally.

Place on a wet paper towel, use rounded forceps to scrape along the mucosa, removing mucous, bacteria, etc.

Place in a 50 mL conical tube with PBS, invert tube a few times.

Pour of the supernatant, refill with fresh PBS.

Repeat the PBS washes 5-6 more times until there is no visible debris.

Cut the intestine in large fragments.

Place the fragments in a 15 mL conical tube with 5-10mL cell dissociation solution.

To make 120mL of cell dissociation solution (adjust as necessary):

A	B
HBSS	114.5 mL
0.5 M EDTA	1.2 mL
1 M HEPES	1.2 mL
FBS	3.12 mL

Incubate for 100rpm on the rotator.

Vortex well for 0h 0m 25s, take out the supernatant with a metal strainer, keep the pieces, discard the supernatant.

Repeat steps 10-12.

Collect the fragments, rinse in HBSS in a small petri dish, cut using a razor blade (~1 mm²).

Digest for 0h 20m 0s at 37°C with slow rotation in 5mL digestion mix.

To make 180mL of digestion mix (adjust as necessary):

A	B
RPMI	126.6 mL
FBS	9 mL
DNase I	720 ul
Dispase (5U/mL)	18 mL
Collagenase D (Roche)	360 ul

From 500mg/mL stock solution, to make working conc of 1mg/mL.

Vortex well for 0h 0m 30s.

Collect the supernatant by filtering through 100 µm strainer in 50 mL falcon tube at Room temperature to avoid cold temperature shock. Keep it On ice afterwards.

Put remaining tissue fragment back into the same tube with 5mL of new digestion mix.

Repeat steps 16-18 two more times.

Combine all appropriate supernatants.

Centrifuge at 3000rpm,4°C.

Prepare Percoll (adjust amounts as necessary).

a. 100% Percoll = `45mL` stock Percoll + `5mL` 10X PBS (`50mL` total).

b. 80% Percoll =`24mL`  100% Percoll +`6mL`  10% RPMI-FBS `30mL`  total).

c. 40% Percoll `20mL`   100% Percoll `30mL`   10% RPMI-FBS`50mL`(  total).

Add 5mL of 80% Percoll to the bottom of a 15 mL conical tube.

Resuspend with LPL cells in 1mL of 40% Percoll to get homogenous solution, then add the remaining 9mL.

Gently add 10mL of the 40% Percoll cell solution on top of the 80% Percoll in the tube.

Centrifuge at 2500rpm, without brake (accel 1, decel 0).

Collect and discard the top layer of epithelium and debris.

Collect white cell layer at the interface between the 40% and 80% Percoll.

Dilute with 10% RPMI-FBS, invert a few times to mix well.

Pellet by centrifugation for 2000rpm,4°C.

Aspirate SN and resuspend cells FACS buffer, then start FACS stain.