Anti cd80 antibody

BMDCs were generated from the bone marrow of 10 week old BALB/c mice. 7 × 10⁵ BMDCs were seeded in 12‐well plates and cocultured with HP, FHP‐1, and FHP‐2 pretreated CT26 cells for 24 h. After incubation, the cells were collected and blocked with antimouse CD16/32 antibody (Biolegend, CA, USA) for 10 min. Subsequently, the cells were stained with anti‐CD11c antibody, anti‐CD40 antibody, anti‐CD80 antibody, and anti‐CD86 antibody (Biolegend) for 30 min. The stained cells were analyzed by flow cytometry (FACS Fortessa, BD Biosciences).

To generate bone-marrow-derived DCs, we isolated bone marrow cells from the femurs of C57BL/6J mice and cultured the cells at 37°C for 7 days with 100 ng/mL human Fms-related tyrosine kinase 3 ligand (PeproTech, Rocky Hill, NJ, USA). Cells were seeded at a density of 1 × 10⁵ cells/well in a 96-well flat-bottomed culture plate (Nunc, Roskilde, Denmark) and were cultured in complete RPMI medium (RPMI 1640 supplemented with 10 vol. % fetal calf serum, penicillin, and streptomycin). These cells were stimulated with CpG ODN or with each LNP-CpG for 24 h. Supernatants were subjected to ELISA to determine the levels of IFN-α (InvivoGen) and interleukin (IL)-12 p40 (BioLegend, San Diego, CA, USA), in accordance with the manufacturers' instructions. To check the levels of co-stimulatory molecules on DCs, we incubated the cells with anti-mouse CD16/CD32 antibody (BioLegend), anti-CD11c antibody (BioLegend), anti-CD11b antibody (BioLegend), anti-CD80 antibody (BioLegend), or anti-CD86 antibody (BioLegend). Then the cells were analyzed by means of flow cytometry (NovoCyte Flow Cytometer, ACEA Biosciences, San Diego, CA, USA).