Kits for intracellular staining

Colon lamina propria (LP) cells were isolated as described previously and stained for flow cytometry (26 (link)). Briefly, the colon tissues were washed and cut into 1–1.5 cm sections incubated in Hanks' Balanced Salt Solution (Sigma-Aldrich, St. Louis, MO) with 5 mM 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 1 h 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: CD3 (clone 145-2C11), CD4 (clone GK1.5) from Biolegend (San Diego, CA), CD45.2 (clone 104) from BD Biosciences (San Jose, CA), FoxP3 (clone FJK-16s), RORγt (clone B2D) from eBioscience (San Diego, CA). The cells were fixed and permeabilized using kits for intracellular staining (eBioscience) and the manufacturer's instructions. All data were collected on a BD Fortessa LSRII (BD Biosciences) and analyzed with FlowJo software (TreeStar, Ashland, OR).

T cells were sorted and seeded in a 96-well plate. Stimulating the cells with PMA (25 ng/ml) to analyze the intracellular INF-γ. Stimulating with PMA (25 ng/ml) and ionomycin (500 ng/ml) to analyze the intracellular TNF-α. 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 IL-17A intracellular cytokine staining. For IL-22 production, adding IL-23 (40 ng/ml, R&D Systems, Minneapolis, MN), PMA, ionomycin, and Brefeldin A into cultures for 4 h. Cells were cultured at 37°C in DMEM medium supplemented with 20% FBS for 4 h. Following the manufacturer’s instructions, cells were fixed and permeabilized with kits for intracellular staining (eBioscience). All data were collected on a BD Fortessa LSRII (BD Biosciences) and analyzed with FlowJo software (TreeStar, Ashland, OR).