Methylated bsa mbsa

Mice were injected with 0.1 mg zymosan intraperitoneally and dosed with 20 mg/kg naproxen in drinking water from day 6 to day 21 or 28. On day 14, mice were injected intraperitoneally with 10 mg/mL methylated BSA (mBSA) (Sigma). Additionally, naive mice were injected with 10 mg/mL mBSA in complete Freund’s adjuvant subcutaneously (Sigma) and left for 10 days. Bone-marrow-derived dendritic cells (DCs) were generated as previously described, and 60,000 per well were incubated overnight with 100 ng/mL LPS and 20 ng/mL Met BSA. DCs were washed and incubated with 300,000 lingual lymph node cells in RPMI (Life Technologies) with 30 U/mL IL-2 (Miltenyi Biotec) for 4 days. Cells were stained for FACS with CD11b, F480, CD11c, MHCII, CD19, CD3, CD4, CD8, and Ki67.

Mice were immunized with methylated BSA (mBSA; Sigma-Aldrich) as described previously (18 (link), 21 (link)). Some mice received 20 × 10⁶ ACs or aACs i.v. for 3 consecutive days after immunization. Other groups of mice received 1.5 × 10⁶ DCs at the time of immunization. In some experiments, neutralization of IL-6 occurred at the time of AC transfer with i.p. injection of 100 μg LEAF purified anti-mouse IL-6 Ab (BioLegend), or control mice were treated with rat IgG1 isotype control (BioLegend). Clinical score was determined by the degree of limping, where 0 = normal walking, 1 = mild limping, 2 = severe limping, and 3 = unable to put weight on leg (18 (link)). Day 3, 6, or 7 post intra-articular injection, inguinal draining lymph nodes and spleens were removed and stimulated with anti-CD3 mAb (0.1 ng/ml) for 72 h. Supernatants from cultures were tested for IL-17A and IL-10 by ELISA (R&D Systems, Minneapolis, MN). In some experiments, ACs or aACs were transferred on the day of immunization only, and spleens were harvested and analyzed 48 h later.

Deoxyribonucleic acid, sodium salt, from Escherichia coli strain B; calf thymus DNA; incomplete Freund's adjuvant (IFA); proteinase K; lysozyme; and methylated BSA (mBSA) were purchased from Sigma-Aldrich (St Louis, MO). Novagen pig genomic DNA was purchased from MilliporeSigma (St Louis, MO). Genomic Maxi-tips, Genomic DNA Buffer sets, and RNase A were purchased from Qiagen (Germantown, MD). TE buffer, pH 8.0, was purchased from Amresco Life Science/VWR (Radnor, PA). A 200-bp DNA ladder (New England Biolabs), 96-well Maxisorp microplates, and T-PER (Tissue Protein Extraction Reagent) were purchased from New Thermo Scientific (Waltham, MA). Goat anti-mouse IgG (H + L) conjugated to horseradish peroxidase was purchased from Novus Biologicals (Littleton, CO). TMB substrate (BD OptEIA Reagent) was purchased from BD Biosciences (San Jose, CA). A ProcartaPlex Mouse Cytokine/Chemokine Panel 1A 36plex Kit was purchased from Invitrogen (Carlsbad, CA).