Complete freund adjuvant (cfa)

Two group of healthy BALB/c mice, five mice per group, were immunized s.c. with 100 μg of Ovalbumin (OVA; Sigma–Aldrich) diluted in 20 μl of saline buffer and emulsified with 30 μl of complete Freud’s adjuvant (CFA; BD BioSciences). The OVA antigen was given twice with 10 days interval, and one group of mice was immunized with 10 μg TcRpL7aRep diluted in 100 μl of saline buffer, 24 h before each OVA immunization. Sera were collected at days 7 and 17 after the first immunization, and anti-OVA antibodies were determined by ELISA using plates coated with 1 μg of OVA antigen/well as described before. Hypersensitivity response was elicited in both group of mice by injecting 30 μl of heat aggregated OVA (HAO) in one rear footpad and saline buffer in the other. Footpad swelling was measured 24, 48, and 72 h after HAO injection and was compared with the basal footpad thickness. HAO was prepared by heating a 2% OVA solution in PBS at 70°C for 1 h. After cooling slowly at room temperature, the antigen was centrifuged at 225 × g for 10 min at 4°C, then was washed twice with PBS and resuspended to the original volume in PBS, as described elsewhere (Titus and Chiller, 1981 (link)).

To induce EAU, B10RIII mice were injected subcutaneously with 100 μL of 20 μg R161 peptide, suspended in phosphate-buffered saline (PBS) and emulsified in Freund’s complete adjuvant (CFA, 1:1 v/v, BD Pharmingen, San Diego, CA, USA), containing Mycobacterium tuberculosis strain H37RA (2.5 mg/mL, BD Pharmingen). The mice were scored by fundoscopy and histologic examination in accordance with the previously described protocols [12 (link)].

Animal experiments were approved by the local state authorities (Landesamt fuer Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen). Rats were housed under specific pathogen-free conditions in the animal research facility of the University of Duesseldorf.
To induce active EAN, female Lewis rats (8 weeks, Janvier, Le Genest-Saint-Isle, France) received subcutaneous injections of 200 μg of P2_55–78 (JPT peptides, Berlin, Germany) in complete Freund’s adjuvant (CFA; BD, Heidelberg, Germany) containing heat-inactivated mycobacterium tuberculosis H37RA (2 mg/ml) (BD). A modified EAN score [9 (link)] was applied: 0 no impairment, 1 reduced tail tone, 2 limp tail, 3 absent righting reflex, 4 gait ataxia, 5 mild paraparesis, 6 moderate paraparesis, 7 severe paraparesis or paraplegia, 8 tetraparesis, 9 moribund, and 10 death due to neuropathy.

12-week-old mice were immunised with 100 µg of bovine collagen type II (bCII) in 100 µl of a 1:1 emulsion with CFA (BD) and PBS intradermally at the base of the tail. Mice were challenged at day 35 with 50 µg of bCII in 50 µl of IFA (BD) emulsion. Mice were monitored for arthritis development as described in⁵⁷ . In short, each visibly inflamed (i.e. swollen and red) ankle or wrist was given 5 points, whereas each inflamed knuckle and toe joint was given 1 point each, resulting in a total of 60 possible points per mouse and day.