Anti ia ie fitc

The monoclonal antibodies for the characterization of DC and lymphocytes by flow cytometry were the following: anti-CD3 FITC, anti-CD4 APC, anti-CD8 PE, anti-CD137-biotin, anti-CD11c APC, anti-CD40-PE, anti-CD86-PE, anti-CD80-PE, anti-F4/80-Cy5 and anti-Ia/Ie-FITC from Biolegend (San Diego, CA, USA). The murine melanoma cell line B16-F10 with haplotype H-2Kb was purchased from The American Type Culture Collection, USA. RPMI-1640 culture medium was purchased from Biowest (Nuaille, France). Ge from bovine skin with a protein percentage of 78% and a water content of 8% was used. HA sodium salt from Streptococcus equi, both from SIGMA (St Louis, MO, USA) with a solubility of 5 mg/mL and an MW of ~1.5–1.8 × 10⁶ Da was used.

Bone marrow-derived dendritic cells (BMDCs) were derived from bone marrow washed from the rear limbs of C57BL/6 mice. Bone marrow cells were strained, washed, and RBCs lysed. The remaining cells were cultured in cIMDM +5% FCS with 20 ng/mL recombinant murine granulocyte macrophage colony stimulating factor (mGM-CSF) (ProSpec, New Jersey, USA), and incubated for 6 days at 37 °C + 5% CO₂. BMDCs were harvested on day 6, plated at 1 × 10⁶ cells/mL in 200 μL cIMDM +5% FCS per well of a round-bottom 96-well plate, and treated with 10 μL of assigned treatments. Treatments included CPBS, CPBS with 10 μg CpGs pre- or post-dialysis, 1 μg lipopolysaccharide (LPS), and 1 mg/mL VP60 VLP with or without CpGs pre- or post-dialysis. Plates were incubated for 24 h at 37 °C + 5% CO₂, and harvested for staining. Harvested BMDCs were stained with near-IR live/dead stain, labelled with anti-CD11c/APC (Clone N418), anti-CD40/PE-Cy7 (Clone 3123), anti-CD80/Pacific Blue (Clone 16-10A1), anti-CD86/PE (Clone GL-1) and anti-I-A/I-E/FITC (Clone M5/114.15.2) (BioLegend, California, USA), and fixed in 2% PFA. FACS analysis was performed on a Gallios flow cytometer, with data analysed using Kaluza version 1.2. The gating strategy is provided as Additional file 2: Figure S2.