Incomplete freud s adjuvant

CIA is a well-established mouse model for human RA [13 ]. Arthritic mice develop swollen joints, chronic inflammation, and joint destruction. CIA was induced by injecting a type II collagen at 2 mg/mL (Chondrex, Washington, USA) and complete Freud’s adjuvant (Sigma-Aldrich, Mannheim, Germany) 1:1 emulsion (200 μL) at the base of the tail in 10-week-old male WT and E2f2^−/− mice. A type II collagen and incomplete Freud’s adjuvant (Sigma-Aldrich) 1:1 emulsion was injected (200 μL) at the base of the tail in 14-week-old mice. Mice were monitored once per day for symptoms of arthritis.

All animal studies were ethically reviewed and performed in accordance with European directive 2010/63/EU and were approved by the Czech Central Commission for Animal Welfare. Female BALB/c mice were immunized on day 0 with a subcutaneous injection of 100 μg protein in complete Freud’s adjuvant (Sigma-Aldrich, St. Louis, MO, USA) (100 µL protein + 100 µL adjuvant), and on days 21, 42 and 62 with an intraperitoneal injection of 50 μg (100 µL protein + 100 µL adjuvant) protein in incomplete Freud’s adjuvant (Sigma-Aldrich). Spleens were harvested on day 64. Anti-nsp10/nsp16 antibodies-producing mouse splenocytes were fused with myeloma cells and the candidate hybridomas were selected using the commercial service of the Monoclonal Antibodies and Cryobank facility at the Institute of Molecular Genetics of the Czech Academy of Sciences.

Female BALB/c mice (age: 4 weeks) were intraperitoneally injected with 100 µg of peptide emulsified in complete Freud’s adjuvant (Sigma). These mice were also each injected with 100 µg of peptide emulsified in incomplete Freud’s adjuvant (Sigma, St. Louis, USA), with a 2-week interval between administrations. The final intraperitoneal injection with saline was administered 4 days before the fusion. Fusions were performed using conventional methodology (30 ). Briefly, spleen cells (1 × 10⁸) obtained from immunized rabbits and the fusion partner Sp2/0 were fused at a ratio of 5:1 with 50% polyethylene glycol 1500 (Roche) at 37°C in serum-free medium. The cells were seeded in 96-well microtiter plates (~2 × 10⁵ spleen cells per well) in hypoxanthine-aminopterin-thymidine medium supplemented with 15% fetal bovine serum. Supernatants were tested for the presence of antibodies specific for the immunogen using ELISA. Hybridomas were cloned by limiting dilution in 96-well microtiter plates.