Isolation and Characterization of Intestinal CX3CR1+ Cells

Following bacterial challenge, luminal contents were carefully recovered by gently flushing the intestine with PBS. Intraluminal CX₃CR1^+/gfp cells were isolated and characterized by flow cytometry as described in details elsewhere (5 (link)). Samples were analysed by BD FACSAria II (BD Biosciences). The following antibodies were used: CD11c (HL3) (BD Biosciences), CD103 (M290) (BD Biosciences), CD103 (2E7) (eBioscience), F4/80 (BM8) (eBioscience), MHC II (M5/114.15.2) (eBioscience), SiglecF (E50-2440) (BD Biosciences). For the isolation of CX₃CR1⁺ cells intestinal tissue from CX₃CR1^+/gfp mice were collected and tissues repeatedly treated with HBSS containing EDTA (2mM). After each treatment tissues were shaken and supernatant discarded. After each wash an aliquot from the supernatant was analysed by microscopy to detect the presence of IECs; EDTA treatment was stopped (usually after 3-4 treatments) when epithelial cells were not present in the supernatant. Tissues were then treated for 50 minutes in RPMI 1640 with 10% FCS, 0.24mg/ml collagenase VIII (Sigma) and 40 U/ml DNase I (Roche) as described by others (19 (link)) ; after shaking cells suspensions were filtered and then purified by gradient separation as described before (5 (link)). Cells were sorted (>95% purity), suspended in PBS and injected into the intestinal lumen for pathogen exclusion assay.