Isolation and Analysis of Colon LP Cells

Colon LP cells were isolated as described previously and stained for flow cytometry (20 (link)). Briefly, the whole colon was washed, cut into 1-1.5 cm sections, incubated twice in Hanks' Balanced Salt Solution (HBSS, Sigma-Aldrich, St. Louis, MO) with 5mM EDTA at 37°C and then digested in RPMI-1640 containing 1 mg/ml collagenase type 1 (Worthington, Lakewood, NJ) and 10% FBS at 37°C for 1h in a shaking incubator. The cells were collected from the interface of 40/80% Percoll gradients (Sigma-Aldrich). The fluorescent dye conjugated-antibodies listed below were used for flow cytometry: Anti-CD3 (145-2C11), CD4 (GK1.5) (Biolegend, San Diego, CA), Thy1.2 (30-H12), CD45.2 (104) (BD Biosciences, San Jose, CA), FoxP3 (FJK-16s), RORγt (B2D), IL-22 (IL22JOP), IL-17A (TC11-18H10) (eBioscience, San Diego, CA). For intracellular cytokine staining, cells were stimulated with PMA (0.1 μg/ml, Sigma-Aldrich), ionomycin (0.5 μg/ml, Sigma-Aldrich) in the presence of Brefeldin A (10 μg/ml, Sigma-Aldrich) for 4 h. For IL-22 intracellular staining, mouse recombinant IL-23 (40 ng/ml, R&D systems, Minneapolis, MN) was added to the PMA, ionomycin and Brefeldin A cultures for 4 h. The cells were fixed and permeabilized using kits for intracellular staining (eBioscience) according to the manufacturer's instructions. All data were collected on a BD Fortessa LSRII (BD Biosciences) and analyzed with FlowJo software (TreeStar, Ashland, OR).