Pe cy7 anti cd8α

Isolated PBMCs were stained in duplicate with FITC-anti-CD3 (BD Biosciences, San Diego, CA, USA), PerCP-Cy5.5-anti-CD19 (BD Biosciences), BV421-anti-CD161 (BD Biosciences), APC-anti-TCRVα7.2 (BioLegend, San Diego, CA, USA), PE-CF594-anti-TCRγδ (BD Biosciences), PE-Cy7-anti-CD8α (BD Biosciences), and PE-anti-CD8β (BD Biosciences) antibodies at 4 °C for 30 min in the dark. Isotype-matched control antibodies were used as negative controls. The frequencies of various T-cell subsets were determined by flow cytometry analysis using the FACSAria II (BD Biosciences) and FlowJo software (v7.6.2; TreeStar, San Carlos, CA, USA).

Cells were stained with: FITC anti-CD8α, FITC anti-CD8β, FITC anti-CD45RB, PE anti-CD45.1, PE anti-CD28, PE anti-CD122, PE anti-CD4, PE anti-CD44, PE anti-CD25, PE-Texas Red (ECD) anti-CD4, PECy5 anti-CD62L, PECy5 anti-TCRβ and PECy7 anti-CD8α (BD Pharmingen, San Jose, CA) for surface markers. Gating strategies for FACS analysis are shown in Additional file 2: Figure S2. For BrdU incorporation assays, cells were stained with Biotin anti-BrdU, FITC streptavidin and Biotin Mouse IgG1, κ isotype control (Biolegend, San Diego, CA). For intracellular cytokine staining, IEL were stimulated with PMA (0.1 μg/ml, Sigma), ionomycin (0.5 μg/ml, Sigma) and Brefeldin A (10 μg/ml, Sigma) for 6 h, fixed with 4% paraformadehyde (Sigma-Aldrich), permeabilized with 0.1% saponin (Sigma-Aldrich), and stained with FITC anti-IFNγ, PE anti-IL-17A, or the FITC/PE labeled Rat IgG1 isotype controls (BD Pharmingen). Flow cytometry was done on a FC500 bench top cytometer (Beckman Coulter, Brea, CA) and the data was analyzed with FlowJo 7.6.5 software (TreeStar, Ashland, OR).