Isolation of colonic and splenic immune cells

Lamina propria mononuclear cells (LPMCs) were isolated as previously described with minor modifications (31 (link)). Briefly, colons were cut longitudinally, washed in Hank’s balanced salt solution (HBSS) supplemented with 1% fetal bovine serum (FBS). Tissues were cut into 0.5-cm² pieces and incubated at 37°C for 20 min in HBSS containing 3 mM ethylenediaminetetraacetic acid and 1mM dithiothreitol to remove epithelial cell. After the incubation, epithelial cells in supernatant were discarded by passing through a 100-µm cell strainer. Then, the remaining pieces was minced and incubated with 1× minimum essential medium–α containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% FBS, and supplemented with 1 mg/ml collagenase IV (Biosharp, China), 1 mg/ml dispase II (Roche, Basel, Swiss), and 0.5 mg/ml deoxyribonuclease I (Roche, Basel, Swiss) for 60 min in a shaking incubator at 37°C. The resulting cell suspension was filtered through a 70-μm cell strainer (BD, USA) and LPMCs were collected after centrifugation at 300g for 10 min.
For splenocyte isolation, mouse spleens were cut into 3- to 5-mm pieces, and smashed using a syringe plunger and then filtered through a 70-μm cell strainer. Red blood cells were removed using red blood cell lysing buffer (Sigma, USA). Prepared single-cell suspensions were further processed flow cytometry analysis.