Preparation of the Single-Cell Suspension and Flow Cytometry

Cell suspensions from mouse spleen were prepared as previously described (29). Cell suspensions were prepared from mesenteric lymph nodes (MLNs) by mechanically disrupting MLNs, and colonic epithelial cell suspensions were prepared by digesting colon tissues with 1 mM EDTA and 1 mm dithiothreitol (30). Single-cell suspensions were obtained by filtering the aforementioned cell suspensions through a 75 μm 200 mesh filter (24).

Splenic or MLN cells were stained with the following fluorescent dye-conjugated antibodies (eBioscience, San Diego, CA, USA) for 30 min at 4°C: APC-F4/80 (Cat# 17-4801), PE-CD11b (Cat# 12-0122), FITC-Gr1 (Cat# 11-6041), FITC-CD4 (Cat# 11-0041), APC-IL-17 (Cat# 17-7177), APC-CD25 (Cat# 17-0251) and PE-Foxp3 (Cat# 12-4771). For the analysis of tmTNF-α expression, NCM460 cells, HCoEpiC cells or colonic epithelial cells were stained with a monoclonal antibody against tmTNF-α (31) for 30 min at 4°C, followed by a FITC-conjugated secondary antibody (FeiYi, Wuhan, China, Cat# ZF-0312). The stained cells were analyzed using an LSRII flow cytometer (Becton Dickinson, San Jose, CA, USA).