Gating strategy for the enumeration of blood DC. Top left: In a forward scatter (FSC)/side scatter (SSC) dot plot with all measured cells a region P1 is drawn to exclude debris. Top right: In a SSC/PE-Cy5 dot plot gated on the P1 population a region P2 is drawn to exclude CD19 positive B cells, CD14 positive monocytes and the granulocytes (with high SSC properties). Down left: In a FL-1/FL-2 dot plot with the cells of population ‘P1 and P2’ a region is drawn around the BDCA-1-PE positive MDC1 cells (P3). Additionally, a region P4 defines the BDCA-2-FITC positive PDCs. Down right: With the cells of population ‘P1 and P2’ a dot plot FL-1/FL-4 is created to define a region with the BDCA-3 strongly positive MDC2s (P5). An isotype control staining involved in the kit helps to set regions in an optimal way. The sum of all blood DC was defined as ‘P1 and P2 and (P3 or P4 or P5)’.