Isolation of lamina propria \(LP) cells from small intestine:
1. Remove small intestine between stomach and cecum. Keep the intestine in a 50ml tube with wash intestine buffer \(WIB) on ice, try to remove gentlyadditional tissue or fat.
2. Flush gut with 10ml cold WIB using gavage needle.
3. In petri dish with WIB remove remaining mesenteric tissue and fat with forceps, using light from a Rx projector.
4. Locate remaining Peyer’s patches \(PPs). Excise by pinching PPs with forceps and cutting as close as possible with scissors.
5. Use flat part of scissors to gently expel remaining mucus and feces.
6. In new petri dish containing WIB open intestine along the length with scissors.
7. Cut gut into small pieces \(5mm) in to a 50ml conical tube with 20ml of cold WIB.
8. Invert several times, vortexing and pour off supernantant. Refill tube and repeat 2 more times.
9. Transfer gut pieces to 50ml falcon tubes containing 20ml pre warm \(37oC) intestinal EDTA buffer.
10. Vortex and Incubate with shaking platform for 20 minutes at 37oC
11. Vortex tube on high for 15 seconds.
12. Allow pieces to settle. Discard supernatant in waste \(this supernantant contains intraepithelial lymphocytes).
13. Add 20ml of intestinal EDTA buffer and repeat steps 10-12 at least twice until clear.
14. Put pieces of tissue in a gauze and wash pieces with WIB twice. EDTA will inhibit collagenase so you must be sure to remove remaining EDTA beforeproceeding.
15. Transfer pieces to 50ml falcon tubes containing 20ml digestion buffer and add 5mg of collagenase 4 \(Sigma) and 0.5mg of DNaseI \(Roche). CAUTION:Concentration of collagenase 4 can be variable even between lots. A titration should be done to determine the optimal concentration to use withoutimpacting cell viability.
16. Incubate in water bath 1 hr at 37oC. Vortex tube every 15 minutes \(until the tissue disappear).
17. Pour supernatant through 70um nylon filter in to 50ml tube.
Once LP cells are isolated, we proceed to do staining in four main steps: 1. viability staining, 2. surface marker staining, 3.fixation/permeabilization and 4. intracellular staining, as follows:
CAUTION: You should use PBS only \(without proteins) as a buffer during viability staining.
1. Wash the cells with 5 ml of PBS
2. Spin at 1200 rpm for 10 min at 4 °C
3. Repeat steps 1 and 2
4. Resuspend cells \(1-10 million) in to 1ml of PBS
5. Add 1ml of Aqua and mix well
6. Incubate at 4 °C for 30 min, protected from the light
7. Add 1 ml of PBS to wash the cells
8. Spin at 1200 rpm for 10 min at 4 °C
9. Repeat steps 7 and 8 once
- Surface staining
10. Transfer cells into a 96 V well plate. Ensure that you divide cell suspensions into two wells \(specific and isotype control staining) per sample.Also, you will need some wells for unstained control, Aqua staining only and fluorescence minus one \(FMO) controls for IgA, B220 as well as CD11cstains. For some intra-cellular stains, isotype controls are “stricter” than FMO. These were used to gauge the level of TNF/iNOS staining.
11. Add 50ml/well of the following surface antibody cocktail \(prepare the cocktail in FACS Buffer):
rat anti-mouse CD16/CD32 \(Fc Block Clone:2.4G2)
12. Incubate at 4 °C for 30 min, protected from the light
13. Wash 2x in FACS Buffer, spin at 1200 rpm for 5 min at 4 °C
14. Resuspend cells in 100 ml/well Cytofix/Cytoperm
15. Incubate for 20 min at 4 °C
15. Wash 2x in 1x Perm/wash \(diluted in water)
16. Resuspend in 50 ml of intracellular staining cocktail \(prepare the cocktail in Perm/wash buffer):
CAUTION: Spin the cocktail 5000 rpm for 10 min before you use it.
N/A \(FMO used)
AF647 Mouse IgG1 Isotype
PE Rat IgG1 Isotype
17. Incubate for 20 min at 4 °C
18. Wash 2x in 1x Perm/wash \(diluted in water)
19. Re-suspend cells in 200 ml of FACS buffer.
20. Store at 4˚C until ready to run FACS.
21. Acquire samples in a LSRII machine. We use BD Compensation beads for compensation as well as application settings to enhance reproducibilitybetween experiments.