Jul 16, 2023

Public workspaceGut prep of intestinal immune cells

DOI
dx.doi.org/10.17504/protocols.io.ewov1q42ogr2/v1
  • 1Columbia University
Connor Monahan
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DOI: dx.doi.org/10.17504/protocols.io.ewov1q42ogr2/v1
Protocol Citation: Connor Monahan 2023. Gut prep of intestinal immune cells. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1q42ogr2/v1
Manuscript citation:
Ivanov, II, McKenzie, B.S., Zhou, L., Tadokoro, C.E., Lepelley, A., Lafaille, J.J., Cua, D.J., and Littman, D.R. (2006). The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126, 1121-1133.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 12, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 84893
Keywords: gut, lamina propria, immune cell, small intestine, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: 0375
Abstract
This protocol details the procedure of purification of immune cells from the mouse small intestine.
Attachments
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786-1999.pdf
68KB
Materials
Materials

  • PBS
  • paper towel
  • conical tube
  • HBSS
  • EDTA
  • HEPES
  • FBS
  • RPMI
  • DNase I
  • Dispase (5U/mL)
  • Collagenase D (Roche)
  • falcon tube
  • Percoll
Procedure
Procedure
57m 55s
57m 55s

Note
Note: This protocol is adapted from Ivanov et al, 2006.
Cut out the entire small intestine – from stomach to caecum.
Note
As you are cutting it out, remove residual fat and connective tissue.

Place in ice cold PBS, move on to the next mouse.
Continue removing all fat and connective tissue.
Cut out the Peyer’s patches.
Note
There should be 9-12 PPs in total.

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.
Wash
Cut the intestine in large fragments.
Place the fragments in a 15 mL conical tube with Amount5-10 mL cell dissociation solution.
To make Amount120 mL of cell dissociation solution (adjust as necessary):
AB
HBSS114.5 mL
0.5 M EDTA1.2 mL
1 M HEPES1.2 mL
FBS3.12 mL

Incubate for Shaker100 rpm, 37°C, 00:10:00 on the rotator.

Incubation
Vortex well for Duration00:00:25 , take out the supernatant with a metal strainer, keep the pieces, discard the supernatant.

25s
Mix
Repeat steps 10-12.
Collect the fragments, rinse in HBSS in a small petri dish, cut using a razor blade (~1 mm2).
Digest for Duration00:20:00 at Temperature37 °C with slow rotation in Amount5 mL digestion mix.

20m
Digestion
To make Amount180 mL of digestion mix (adjust as necessary):
AB
RPMI126.6 mL
FBS9 mL
DNase I720 ul
Dispase (5U/mL)18 mL
Collagenase D (Roche)360 ul
From Amount500 mg/mL stock solution, to make working conc of Amount1 mg/mL .
Vortex well for Duration00:00:30 .
30s
Mix
Collect the supernatant by filtering through Thikness100 µm strainer in 50 mL falcon tube at TemperatureRoom temperature to avoid cold temperature shock. Keep it TemperatureOn ice afterwards.
Put remaining tissue fragment back into the same tube with Amount5 mL of new digestion mix.
Repeat steps 16-18 two more times.
Combine all appropriate supernatants.
Centrifuge at Centrifigation3000 rpm, 4°C, 00:10:00 .

10m
Centrifigation
Prepare Percoll (adjust amounts as necessary).
a. 100% Percoll = Amount45 mL stock Percoll + Amount5 mL 10X PBS (Amount50 mL total). b. 80% Percoll = Amount24 mL 100% Percoll + Amount6 mL 10% RPMI-FBS (Amount30 mL total). c. 40% Percoll = Amount20 mL 100% Percoll + Amount30 mL 10% RPMI-FBS (Amount50 mL total).
Add Amount5 mL of 80% Percoll to the bottom of a 15 mL conical tube.
Pipetting
Resuspend with LPL cells in Amount1 mL of 40% Percoll to get homogenous solution, then add the remaining Amount9 mL .
Gently add Amount10 mL of the 40% Percoll cell solution on top of the 80% Percoll in the tube.
Centrifuge at Centrifigation2500 rpm, 00:20:00 , without brake (accel 1, decel 0).

20m
Centrifigation
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 Centrifigation2000 rpm, 4°C, 00:07:00 .

7m
Centrifigation
Aspirate SN and resuspend cells FACS buffer, then start FACS stain.