Cell culture

The murine macrophage cell line Raw264.7, the human intestinal epithelial cell line Caco-2, and mouse BMDM were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and penicillin (100 U/ml) and streptomycin (100 mg/ml; P/S). DC2.4 cells were cultured in RPMI 1640 medium with 10% FBS and P/S. Furthermore, 1% nonessential amino acids were supplemented in the complete medium for Caco-2 cells. Raw264.7 cells [macrophage cell line derived from male BALB/c mice, American Type Culture Collection (ATCC) no. TIB71] and Caco-2 cells (human intestinal epithelial cell line derived from white male, ATCC no. HTB-37) were purchased from ATCC (Manassas, Virginia, USA). DC2.4 cells (mouse dendritic cell line derived from C57BL/6 mice, SCC142) were purchased from Merck Millipore (Sigma-Aldrich, USA). BMDM were generated as previously described (65). In brief, bone marrow cells were collected from the male C57BL/6J mice and were cultured in DMEM supplemented with 20% FBS and macrophage colony-stimulating factor (30 ng/ml) for 6 days. The adherent cells were differentiated macrophages, which were confirmed by flow cytometry (F4/80+/CD11b+) or microscopy (immunofluorescence staining with CD68 or F4/80+). Cells were maintained in a humidified incubator with 5% CO2 at 37°C. To establish the intestinal barrier disruption model, 2% DSS (colitis grade, 36 to 50 kDa, MP Biomedicals, USA) and 5 ng/mL TNF-α (Sigma-Aldrich, St. Louis, MO, USA) were used to incubate with Caco-2 cells for 20 h. For cellular inflammation model, Raw264.7 and DC2.4 cells were seeded in plates, and then LPS (100 ng/mL, from Escherichia coli O55:B5, Sigma-Aldrich, St. Louis, MO, USA) was added and incubated for 20 h.