Cd11c hl3

The antibodies with following specificities were used; CD4 (GK1.5, Tonbo), CD8 (53.67, Tonbo), CD11c (HL3, BD), CD11b (M1/70, eBioscience), TCRβ (H57-597, BD), NK1.1 (PK136, eBioscience), γδTCR (GL3, Biolegend), CD44 (IM7, eBioscience), CD122 (TMβ1, BD), CD62L (MEL-14, eBioscience), IL-15Rα (DNT15Rα, eBioscience), IL-2Rα (3C7, Biolegend), γc (4G3, BD), IL-17 (eBio17B4, eBioscience), IFNγ (XMG1.2, Biolegend), pSTAT5 (clone 47, BD), Foxp3 (MF23, BD). Fluorochrome-conjugated CD1d tetramers loaded with PBS-57 were obtained from the NIH tetramer facility (Emory University, Atlanta, GA).

Intestinal tissues were fixed in 4% paraformaldehyde, rehydrated in 20% sucrose, and frozen in OCT media (Sakura). Tissues were cut into 7-8μm sections and treated with ice cold acetone. Sections were treated with biotin-avidin blocking reagent when necessary (Vector labs) and stained with the following biotinylated or directly conjugated antibodies: CD8β (YTS156.7.7, Biolegend), CD4 (RM4-5, eBioscience), CD103 (M290, BD Biosciences), CD90.1 (HIS5.1, eBioscience), Epcam (G8.8, Biolegend), CD11c (HL3, BD Biosciences), B220 (RA3-6B2, eBioscience). Rabbit anti-Yersinia pseudotuberculosis (ab26120, Abcam) and anti-rabbit Dylight 649 (ab96926, Abcam) were used to stain for bacterial antigens. Stained slides were mounted with Prolong Gold antifade reagent (Invitrogen), imaged using a Nikon 90i, and analyzed using Adobe Photoshop software.
The number of OT-I cells/villus was determined by sectioning a ‘Swiss roll’^{52 (link)} of the distal third of the small intesine. Five or more sections/mouse that were at least 400μm apart were stained and imaged. A villus and the underlying submucosa and muscularis were considered a single villus, and the number of OT-I cells in each region was determined. The number of OT-I cells/villus were binned and plotted as the percentage of villi containing a given range of OT-I 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.