Cd103 m290

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.

For intravascular staining, mice were injected intravenously with 3μg of anti-CD45 antibody (BD Biosciences, San Jose, CA, USA). Lungs and spleen were harvested after euthanasia with pentobarbital (250mg/kg). Lymphocytes were isolated by physical disruption of tissue using a GentleMACs machine (Miltenyi Biotec, San Diego, CA, USA) followed by Fico-LITE density gradient centrifugation. Isolated mononuclear cells were washed with PBS and resuspended in FACS staining buffer (PBS + 1%FBS + 0.05% sodium azide). Cells were stained with fluorochrome-labeled antibodies for the lineage markers CD3 (145-2C11) and CD8 (53-6.7) and the phenotypic markers CD44 (IM7), CD62L (MEL-14), CD127 (A7R34), KLRG-1 (2F1/KLRG1), CD69 (H1.2F3), and CD103 (M290) (BD Biosciences or BioLegend, San Diego, CA, USA). The amine viability dye AquaBlue (Invitrogen) was used to identify dead cells. Antigen specificity was determined using D^bM_187-195 tetramers conjugated to APC (MBL, Woburn, MA, USA). All samples were stained with a pre-mixed antibody cocktail for 20 min at 4°C. Data were collected using an LSR II flow cytometer (BD Biosciences) and analyzed with FlowJo software (TreeStar San Carlos, CA, USA).

Cells were stained with antibodies for B220 (RA3-6B2), CD25 (7D4), CD117 (2B8), IgD (11–26c.2a), or CD103 (M290) from BD Pharmingen (San Jose, CA). CD19 (6D5), CD5 (53-7.3), CD138 (281-2), IL-21R (A49), CD40 (3/23), MHCII (M5/114.15.2), CD138 (281-2), CD11c (N418) and PE/Cy7-conjugated anti-mouse IL-10 (JES5-16E3) antibodies were from Biolegend. For intracellular IL-10 staining, cells were stimulated in vitro in the presence of 10 μg/mL LPS (Sigma-Aldrich), 50 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich), 500 ng/ml ionomycin (Sigma-Aldrich) and 2 μM monensin (eBioscience). Cells were stained for surface markers before fixation with 2% paraformaldehyde and membrane permeabilization with 1% saponin. Fluorescence minus one (FMO) was used to determine the gating strategies. To analyze cells, a Cytomics FC500 instrument (Beckman Coulter, Hialeah, FL) was used before further assessment with Flowjo 7.6.1 software (Tree Star, Ashland, OR).

Flow cytometry was performed using a FACSCalibur (BD Biosciences). Data were analyzed using FlowJo (Tree Star Inc, Ashland, OR). and WinMDI software (Scripps Institute, Lo Jolla, CA). The percentage of positive cells for a given marker was based on cutoff points chosen to exclude >99% of the negative control population. Fluorochrome-conjugated mAbs to mouse CD3e (145-2C11), CD4 (GK1.5), CD8a (53-6.7), CD8b (H35-17.2), CD44 (IM7), and CD103 (M290) and the respective species- and isotype matched negative control mAbs were purchased from BD Biosciences.
The presence of donor-reactive antibodies was determined by the ability of sera to bind to DBA/2 splenocytes detected with goat anti-mouse IgG (γ chain specific) antibodies (Southern Biotechnology, Birmingham, AL) measured as mean fluorescence intensity (MFI) of DBA/2 SC targets. Background fluorescence was determined for each subtype by taking the MFI plus three standard deviations of sera from five naïve C57BL/6 mice. Negative controls included SC plus each detection antibody; MFI values ranged from 25 to 35.