Isolation and Flow Cytometry of Dendritic Cell Subsets

DCs were isolated from lymphoid organs as described previously (9 (link)). Briefly, single cell suspensions were prepared from pooled skin-draining lymph nodes and enriched for cDCs using antibody depletion and magnetic bead enrichment. Cells were incubated with anti-mouse monoclonal antibodies CD11c Brilliant Violet (BV) 421, MHC II APC-Cy7, CD8α PE-CF594, XCR1 PE, CD103 FITC, and CD326 PE-Cy7 purchased from BD Biosciences (Franklin Lakes, NJ, USA) or BioLegend (San Diego,CA, USA) to identify DC subsets. DCs were sorted from Propidium Iodide negative events using a FACSAriaIII cell sorter (BD Biosciences). During sorting, DCs were collected in buffer containing FCS (Sigma-Aldrich, St. Louis, MO, USA) and EDTA (Sigma-Aldrich) and kept on ice at all times before being recovered by centrifugation and stored in Trizol reagent (Invitrogen, Carlsbad, CA, USA).