Identification of IFN-α production by intracellular staining of flow-sorted lymph node CD123⁺ pre-DCs after in vivo infection with influenza A virus. Staining of cytospin preparations of Flt3L-mobilized blood CD11c⁺ pre-DC (A) and CD123⁺⁺ pre-DC (B) subsets flow-sorted from peripheral blood of rhesus monkeys 5 days after in vivo infection with influenza A virus shows no evidence of IFN-α production. Panel B inset shows IFN-α production (cytoplasmic red dots) in CD123⁺⁺ pre-DCs mobilized by Flt3L and infected in vitro with HSV type-1 (Kos strain; 10 pfu/cell) as positive control for IFN-α production by DCs). In contrast to peripheral blood, intracellular IFN-α could be readily detected (arrows) in lymph node CD123⁺ pre-DCs (D) but not in CD11c⁺ pre-DCs (C) on day 3 after influenza infection. IFN-α was detected by immunofluorescence staining with biotinylated mouse antihuman IFN-α followed by streptavidin Cy3 conjugate (original magnification × 1000 for all panels).