Heavy paraffin oil

For arthritis induction and T cell proliferation assays, 8 to 10 week old mice were immunized subcutaneously at the base of the tail with 100 μg of peptide or bovine CII emulsified in equal volumes of CFA consisting of 85% heavy paraffin oil (Fisher Scientific), 15% mannide mono-oleate (Sigma), and 4 mg/ml of heat killed mycobacterium (H37Ra, Difco) [43 (link)]. For arthritis studies, mice were examined 3 times per week starting at day 18 after immunization and the presence of arthritis, number of affected limbs, and the severity were assessed. Severity of disease was evaluated by visual inspection and assigned a score using a scale of 0 to 4 based on the degree of inflammation as described [43 (link)].

For arthritis induction and T-cell proliferation assays, 8- to 10-week-old mice were immunized subcutaneously at the base of the tail with 100 μg of bovine CII emulsified in complete Freund’s adjuvant (CFA) consisting of 85 % heavy paraffin oil (Fisher Scientific, Fair Lawn, NJ, USA), 15 % mannide monooleate (Sigma-Aldrich, St. Louis, MO, USA), and 4 mg/ml of heat-killed mycobacterium (Difco H37Ra; BD Diagnostics, Sparks, MD, USA) [27 (link)]. FTY720 (Cayman Chemical, Ann Arbor, MI, USA) was diluted in nitrogen-bubbled 10 % ethanol and injected intraperitoneally. Treatment schemes varied among experiments and are described in the Results section. In some experiments, mice were treated with 250 μg of rat anti-CD25 (clone PC61) or a control rat antibody (MOPC 167) once weekly for 3 weeks. Peripheral blood was monitored by flow cytometry to ensure efficacy in the depletion of the CD25⁺Foxp3⁺ T cells. For arthritis studies, mice were examined three times per week starting at day 18 after immunization, and the presence of arthritis, number of affected limbs, and severity of inflammation were assessed. Severity of disease was evaluated by visual inspection and assigned a score using a scale of 0–4 as described elsewhere [27 (link)].