Anti cd80 pacific blue

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.