FL-DC do not require TLR9 or MyD88 for activation in response to HSV-1. Total FL-DC (2.5 × 106 per ml) generated from WT, TLR9 KO, or MyD88 KO mice were stimulated with HSV-1 (107 pfu/ml), CpG-2216 (1 μM) or R848 (1 μg/ml). (A) The supernatants were removed and tested for IFN-α or IL-6 production by ELISA. (B) Cells were stained with antibodies directed to CD86 or CD40. (C) Total FL-DC (1 × 106 per ml) from WT or TLR9 KO mice were then stimulated for 22 h with a titration of HSV-1 from 1 × 107 – 0.16 × 106 pfu/ml [10 – 0.16 multiplicity of infection (MOI)], and the supernatants were tested by ELISA for IFN-α, TNF-α, or IL-6 production. (D) Sorted pDC and cDC (2.5 × 105 per ml) purified from FL-DC of WT or TLR9 KO mice were stimulated with HSV-1 or CpG-2216. Supernatants were tested by ELISA for IFN-α, TNF-α, or IL-6 production. (E) Sorted pDC or cDC (5 × 105 per ml) from FL-DC of WT mice were stimulated with HSV-1 in the presence or absence of IL-3 and GM-CSF and the supernatants were tested by ELISA for IFN-α. One representative experiment of at least three is shown. Error bars represent the range of duplicate samples; asterisks indicate that cytokines were not detectable by ELISA.