Cpg odn 1826

For BMDC generation, bone marrow from BALB/c mice was harvested from femurs and tibias and a single cell suspension was generated. 8×10⁶ bone marrow cells were seeded in each Petri dish (#FB0875712, Fisher Scientific, Hampton, NH) and cultured in 10 ml T cell medium with GM-CSF (#576306, Biolegend, San Diego, CA) (at 10 ng/mL). On day 3, 10 ml medium was added. On day 5, the medium was refreshed by removing 10 mL culture and replacing with 10 ml fresh medium. On day 6, Poly (I:C) (LMW) (InvivoGen, San Diego, CA) and CpG ODN 1826 (5’-TCCATGACGTTCCTGACGTT-3’) (Integrated DNA Technologies, Inc. San Diego, CA) added to activate the cultured cells. On day 7, the cells were harvested and CD11c⁺ cells were isolated using the CD11c MicroBeads UltraPure for mouse (#130–108-338, Miltenyi Biotec, Bergisch Gladbach, Germany) and LS column (#130–042-401, Miltenyi Biotec, Bergisch Gladbach, Germany). These activated CD11c⁺ cells (*BMDC) were pulsed with the HNT epitope peptide, HA_126–138 (HNTNGVTAACSHE, New England Peptide, Gardner, MA), at the indicated concentrations (1.25 μM, 12.5 μM, 125 μM) for 1hr at 37⁰C. The HNT peptide-pulsed, activated BMDCs are referred to as Ag/*APC.

Single cell suspensions were produced from pooled groups of two spleens, filtered, and incubated with anti-CD11c mAb-conjugated magnetic microbeads (Clone N418; Miltenyi Biotech) for 15 min in the dark at 4°C according to manufacturer’s instructions. Cells were washed and resuspended in cold Automacs buffer and passed through an LS MACS separation column (Miltenyi Biotech). Columns were washed three times with 3 mL of cold Automacs buffer and the positive fraction collected. Enriched cells were plated at 0.4-1 × 10⁶ cells/dish and stimulated for 7 h at 37°C with 1 μg/mL LPS (055:B5; Sigma-Aldrich) or 1 μg/mL CpG (ODN 1826; Integrated DNA Technologies). After 2 h of stimulation, 1 mg/mL of Brefeldin A (BD Biosciences) was added to each dish. After 7 h incubation, intracellular stains for IL-12/IL-23p40 (clone C15.6, Biolegend) and TNF-α (clone MPG-XT22, eBioscience) were performed after surface staining and fixation/permeabilization of the cell membrane using Cytofix/Cytoperm solution (BD Biosciences).