Fixable viability dye 780 apc cyanine 7

Phenotype of moDC, T cells and B cells after coculture was analyzed by flow cytometry. Collected cells were stained with Fixable Viability Dye 780-APC Cyanine 7 (eBioscience, USA), followed by blocking of nonspecific binding sites with human Fc block (BD Biosciences, USA) in PBS containing 1% bovine serum albumin (Roche, Switzerland). Extracellular staining was performed using titrated volumes of the following antibodies: CD11c-PerCP eFluor 710 (clone 3.9), HLA-DR-PE (clone LN3), CD80-FITC (clone 2D10.4), CD86-PE-Cy7 (clone IT2.2), OX40L-APC (clone RM134L), CD4-PerCP-Cy5.5 (clone OKT4), CXCR3-AF488 (clone 1C6/CXCR3), CRTH2-APC (clone BM16), CD19-PE-Cy7 (HIB19), CD4-PE (clone RPA-T4), CD25-AF488 (clone BC96) (purchased from eBioscience or BD Biosciences). Cells were permeabilized with the Intracellular Fixation & Permeabilization Buffer Set (eBioscience, USA) to allow staining with IL13-PE (clone JES10-5A2). Flow cytometric measurements were performed using BD FACS CantoII (Becton Dickinson, USA) and data was analyzed using FlowLogic software, (Inivai Technologies, Australia). Representative gating strategies are given in Supplemental Figure 4.

All cells collected for flow cytometric analysis were transferred to 96-well plates (Costar Corning Incorporated). After washing the cells with PBS, the viability of the cells was determined with Fixable Viability Dye 780-APC Cyanine 7 (eBioscience). Blocking buffer [PBS with 2.5% FCS and human Fc block (BD Biosciences, United States)] or FcR blocking reagent mouse (Miltenyi Biotech, United States) was added for 15 min at 4°C to prevent non-specific binding of antibodies. Murine samples were stained using titrated volumes of the following antibodies: CD4-BV510 (clone RM4-5), CD69-PE-Cy7 (clone H1.2F3), CXCR3-PE (clone CXCR3 473), T1ST2-FITC (clone DJ8), CD25-PerCP-Cy5.5 (clone 3C7), and FoxP3-FITC (clone FJK-16 s). After 30 min of staining at 4°C, stained cells were washed. Cells were resuspended, flow cytometric measurements were performed using BD FACS Canto II (Becton Dickinson, United States), and acquired data were analyzed using FlowLogic software (Inivai Technologies, Australia). A representative gating strategy is shown in Supplementary Figure S2.

After IEC/moDC co-culture, ccDC were collected and stained for flow cytometry analysis using CD11c-PerCP eFluor 710 (clone 3.9), CD14-APC (clone 61D3), HLA-DR-PE (clone LN3), CD80-FITC (clone 2D10.4) and CD86-PE Cyanine 7 (clone IT2.2) (all from eBioscience, San Diego, CA, USA). Viability was determined using Fixable Viability Dye 780-APC Cyanine 7 (eBioscience). Non-specific binding sites were blocked using PBS supplemented with 5% FCS before extracellular antibody staining. Flow cytometry measurements were done using BD FACS Canto II (Becton Dickinson, Franklin lakes, NJ, USA) and data were analyzed using Flowlogic software version 7 (Inivai Technologies, Mentone, VIC, Australia).