Dec205 pe

Draining LNs were resected at indicated time points postsubcutaneous injections of 50 μg of dye-labeled NPs. Single cell suspensions of LNs were made physically with frosted slides. Cells were stained with CD11c-eFluor450, F4/80-FITC, B220-PE, and PDCA-PerCP-eFluor710, all from eBioscience. Cells were then examined with Cyan ADP (Dako) and analyzed with Summit software. For DC subset analysis, LN cells were stained with CD11c-eFluor450, CD8-FITC, and DEC205-PE (eBioscience).

The following reagents were procured as described: CD11c-FITC, CD11c-PE, CD11b-APC, I MHC class II (I-Ad/I-Ed)-FITC, CD40-V421 (BD Bioscience, USA), CD80-FITC, CD86-PE, CD206-PE, CD14-PE, CD115-PE, F4/80-APC, CD103-PE, CD8a-V430, CD197-PE, B220-PE, CD207-PE, DEC-205-PE, CD124-PE, sigH-PE (eBioscience, USA). Blocking antibodies against TRAIL, FasL, TNFR1 (Biolegend, USA), ultrapure mouse rIFNβ, rTNFα (Biolegend, USA). DAPI, Cell Trace Violet (Invitrogen, USA), L-NMMA, β-Mercaptoethanol (Sigma, USA), FeTPPS (Calbiochem, USA), Bx795 (Invitrogen, USA). LPS 055:B5 (TLR tested, Invivogen, USA). LTA-BS, Pam2CSK4, Pam3CSK4, Poly(I:C) HMW, Flagellin-PA Ultrapure, Imiquimod, ODN CpG 1826 (Invivogen, USA). Pharmaceutical grade Immunomax® (IMM) has been purchased from Immapharma Ltd (Russia).