Phenotypic Identification of Migratory DCs

Freshly isolated mesenteric lymph nodes (MLNs) were analyzed by flow cytometry (14 (link)). Identification of migratory DCs in the MLNs was performed by gating CD103+ cells out of CD11c+ MHC-II cells in the MLNs (the gating strategy is shown in Figure 2A). Cells obtained and resuspended in PBS with 1% bovine serum albumin were incubated with antimouse CD16/CD32 (Mouse BD Fc Block; BD Pharmingen) for 20 min on ice to block nonspecific binding sites. For surface staining, cells were incubated with CD4-PerCp-Cy5.5, CD69-PE, CD25-AlexaFluor488, CD11c-PerCp-Cy5.5, CD103-APC, CD40-FITC, CD86-PE-cy7, MHCII-PE, CD3-Percy5.5, CD27-PE, CD19-APC, B220-FITC (eBiosciences). Foxp3-PE-cy7, and Tbet-APC (eBioscience) were used for intracellular staining. Staining and flow cytometry were performed as described previously (14 (link)).