Mouse small intestines were dissected, and fat tissues and Peyer’s patches were removed. The intestines were cut open longitudinally and washed with Dulbecco’s modified Eagle’s medium (DMEM) until no fecal pellets were observed. The intestines were then cut into approximately 5-mm-long pieces. The intestinal pieces were incubated in 37°C prewarmed DMEM containing 3% fetal bovine serum (FBS), 20 mM Hepes, 5 mM EDTA, and dithiothreitol (0.15 mg/ml) for 10 min with constant agitation by droppers in a 37°C water bath. The digested cells that were collected were intraepithelial lymphocytes. Then, the left small intestine was incubated in a solution of 3% FBS, 20 mM Hepes, DNase I (0.125 mg/ml), and collagenase II (0.5 mg/ml) in 37°C prewarmed DMEM for 5 min with constant agitation by droppers in a 37°C water bath, and the dissociated cells that were collected were LP lymphocytes. Last, the collected cells were isolated by passing the tissue through a 200-mesh cell filter membrane, followed by 80%/40% Percoll (GE Healthcare) gradient centrifugation. Cells were carefully removed from the interface, washed with PBS, and resuspended in culture medium for further analysis.

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