Strategy for the isolation and sorting of thymic pDC and mDC. (A) Thymus tissue was digested to create a single cell suspension. Using Nycodenz density centrifugation cells were then divided into a low-density (LDF) and a high-density fraction (HDF). Thymocyte subpopulations were sorted from the HDF. DC were enriched from the LDF by magnetic bead depletion (MACS) and high speed flow cytometric cell sorting (FACSAria). (B) Gating strategy for sorting thymic DC; (i) Viable cells were selected by live gating using forward (FSC) and side scatter (SSC); (ii and iii) Doublets were excluded based on FSC-width (W), -height (H) and SSC-W, H; (iv) HLA-DR-positive, cocktail (CD3, CD15, CD19, GlyA)-negative cells were selected and (v) CD123+ (pDC) and CD11c+ (mDC) populations were sorted. On average, the recovery of isolated DC was 3 × 105 pDC and 2 × 105 mDC per 109 total thymic cells. The purity for pDC and mDC was always greater than 98%.