Intestinal cell isolation

Small and large intestine were excised and freed of mesentery and fat. Feces were removed and PPs were excised from the small intestine. Both small and large intestine were opened longitudinally and washed in HBSS, followed by HBSS + 1 mM DTT (Sigma-Aldrich). Tissue was cut into 1-cm pieces and incubated with 25 ml of HBSS + 5% FBS + 5 mM EDTA for 15 min at 37°C at 230 rpm. Supernatant containing intraepithelial lymphocytes was decanted and spun down (for RNA isolation from epithelial cells, see next section). Tissue was washed with 10 ml of HBSS + 5% FBS for 15 min at 37°C at 230 rpm. The gut tissues were then finely chopped and digested in HBSS + 5% FBS + 1X Sodium Pyruvate + 25 mM Hepes + 50 µg/ml DNaseI + 0.05 mg/ml Collagenase VIII for 1 h at 37°C at 100 rpm. Cells were separated by discontinuous Percoll gradient (70%/35%) centrifugation. Cells were isolated from the interphase.