Isolation of Lymphocytes from Tissues

Single-cell suspensions were prepared from inguinal and mesenteric lymph nodes by mechanical disruption and passed through 100 μm filter (Corning). Colonic lamina propria T cells were isolated, as previously described^{48 (link)}. Briefly, colons were opened longitudinally and contents were flushed with ice-cold Hanks balanced salt solution, HBSS (Cellgro). Each colon was cut into small pieces and washed with HBSS solution supplemented with 5% FCS (HyClone) and 2 mM EDTA at 37 °C. A single-cell suspension was obtained after treatment with Collagenase D (1.0 mg/ml) and DNase I (0.1 mg/ml) (both from Roche). A purified and concentrated suspension of lamina propria lymphocytes was obtained after centrifugation on Percoll (GE Healthcare) gradient (45% and 70%). The interface, enriched in leukocytes, was collected and used for experiments. Lungs and liver were harvested, and lymphocytes were isolated by enzymatic digestion for 20 min, using Collagenase D (1.0 mg/ml) and DNase I (0.1 mg/ml) (both from Roche) at 37 °C. For T cell enrichment, Lymphocyte Separation Medium (Corning) was used. The interphase was collected and used for further analysis.