Dissect the small and large intestines for isolation of intestinal lamina propria cells, place the small and large intestines immediately On ice cold PBS.

Open the intestines longitudinally after removing the mesenteric fat and Peyer’s patches (small intestine), wash out the luminal contents with cold PBS.

Wash the tissue pieces for 0h 10m 0s in 1millimolar (mM) dithiothreitol (DTT)/PBS at Room temperature on a rocker to remove mucus, followed by a wash for 0h 25m 0s in 10millimolar (mM) EDTA/30millimolar (mM) HEPES/PBS at 37°C on a platform shaker (180rpm,0h 0m 0s) to remove epithelium.

After a 0h 2m 0s wash in complete RPMI, digest the tissue in a 6-well plate for 1h 30m 0s in complete RPMI with 150U/ml (small intestine) or 300U/ml (large intestine) collagenase VIII (Sigma) and 150µg/µL DNase (Sigma) in a cell culture incubator (5% CO₂).

Pass the tissue digests through a 100 µm cell strainer and separate them by centrifugation (1200x g,0h 0m 0s for 0h 20m 0s) using a 40/80% percoll gradient.

Collect the immune cells at the 40/80% interface.

For the spleen, pass the tissue through a 100 µm cell strainer and incubate in red cell lysis buffer (Sigma) for 0h 8m 0s at Room temperature.

Wash both spleen and intestine immune cells with 0.5% BSA/PBS before staining and fixation (eBioscience Foxp3 / Transcription Factor Staining Buffer Set).

Use , CD16/32 antibody (eBioscience) for flow cytometry staining to block the non-specific binding to Fc receptors before surface staining.

Isolate the immune cells from intestinal lamina propria and stain with antibodies against the following markers:

A	B
Marker	Stain
CD103	PerCP-efluor710
CD11b	SuperBright645
CD11c	FITC
CD19	FITC
CD3e	PE
CD4	APC
CD45.2	BV421
CD64	APC-Cy7
CD8a	APC-e780
CSF1R	PE
Ly6C	APC
MHCII I-A/I-E	PE or PerCP-efluor710
TCRβ	PerCP-Cy5.5

For some panels, a lineage marker mix (Lin) contained TCRβ, B220, Ly6G and Siglec-F (PE-Cy7).

Discriminate the live and dead cells by Live/Dead Fixable Aqua Dead Cell Stain Kit (Invitrogen).