Dnase

Colon was isolated from NOD mice and opened longitudinally, washed with 1X PBS to remove fecal contents and cut into 0.5 cm pieces. The epithelial cells, mucus, and fat tissue, from the colon, were removed by incubating in 1X HBSS (Sigma-Aldrich, St. Louis, MO, USA) containing 5 mM EDTA (Sigma-Aldrich) and 1mMDTT(dithiothreitol) (BioRad) for 30 min at 37 °C. Colon was then minced and digested using digestion solution containing 1 mg/mL collagenase VIII (Sigma-Aldrich), 0.5 mg/mL DNase (MP Biomedicals, Santa Ana, CA, USA), and 2% FBS (Thermo Scientific) in 10 mL of RPMI (Sigma-Aldrich) for 30 min at 37 °C at 200 rpm. The crude cell suspension was resuspended in 10 mL of 40% Percoll fraction and overlaid in 5 mL of 80% Percoll fraction (MP Biomedicals) and centrifuged at 1000 g for 20 min at room temperature with brake turned off^{41 (link)}. Lamina Propria mononuclear cells (LPMCs) were collected from the white ring at the interphase of the Percoll solution.

HSCs were isolated from male BALB/c mice (14 weeks old) as described previously^{41 (link)} with some modifications. In brief, the livers were perfused in situ with phosphate-buffered saline (PBS) and then with Gey’s balanced salt solution (GBSS) supplemented with collagenase (0.5 mg/ml; Sigma-Aldrich) and pronase (1 mg/ml; Sigma-Aldrich). The perfused livers were dissected, and the attached gall bladders and connective tissues were removed. The liver cell suspensions were further digested in GBSS supplemented with collagenase (0.25 mg/ml), pronase (0.5 mg/ml), and DNase (0.07 mg/ml; MP Biomedicals, Santa Ana, CA, USA), for 12 min in a 37 °C water bath. The cells were then washed and centrifuged in a 13.4% Nycodenz gradient at 1400×g for 20 min without brake. The interface containing the enriched HSCs was collected and washed with GBSS. Then, the isolated HSCs were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. The purity of the HSCs was assessed by microscopic observation. The HSCs were passaged before reaching 70% confluence in the primary culture and used as activated HSCs. The activation status of the HSCs was assessed on the basis of their increased expression of α-SMA and collagen type I as well as through their morphologic changes.