Complete freund s adjuvant cfa

Tert-butanol (t-BuOH), dimethyl sulfoxide (DMSO), diethyl ether, ethyl acetate, acetone, methanol (MeOH), dimethylacetamide (DMA), 2,2′-azobis(isobutyronitrile) (AIBN), 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile) (V70), acetic acid (CH₃COOH), dexamethasone (DEX), 4-(2-oxopropyl)benzoic acid (OPB), N-(3 dimethylaminopropyl)-N′-ethylcarboiimide hydrochloride (EDC), 4-(dimethylamino)pyridine (DMAP), dimethylformamide (DMF), dichloromethane (DCM), and porcine liver esterase were obtained from Merck KGaA (Darmstadt, Germany). The fluorescent dyes cyanine 5.5-succinimidyl ester (Cy5.5-NHS ester) and cyanine 5.5 (Cy 5.5) were obtained from Lumiprobe GmbH, (Hannover, Germany). Milli-Q water (H₂O) was used for all experiments and obtained from the appliance Millipore Merck, Darmstadt, Germany.(resistivity 18.2 MΩ·cm, 25 °C, organic carbon ≤ 5 ppb). dexamethasone (2 mg/mL of excipient QSP) used in in vivo experiments was purchased from Vibrac S.A. (Carros, France) and 3-aminophthalhydrazide monosodium salt was obtained from Thermo Fisher GmbH (München, Germany). Complete Freund’s adjuvant (CFA) was obtained from Becton, Dickinson and Company, Franklin Lakes, NJ, USA.

Female 8- to 10-week-old GFAP^CreSirt1^fl/fl and Sirt1^fl/fl mice were used for EAE induction. Mice were immunized subcutaneously at 2 sites on the back with 200 μg MOG_35–55 peptide (GenScript) emulsified in Complete Freund’s Adjuvant (CFA) (BD Biosciences) supplemented with 4 mg/mL Mycobacterium tuberculosis H37Ra (BD Biosciences); 200 ng pertussis toxin (MilliporeSigma) was injected intraperitoneally into each mouse on day 0 and day 2 p.i. All mice were monitored for weight and clinical signs daily until 25 or 30 days after induction of EAE. Mice were euthanized if they showed a 20% loss of maximum body weight. Gel food was supplied at the onset of EAE disease. Clinical scores were recorded on the following scale: 0, no clinical signs; 1, limp tail; 2, limp tail with weak/partially paralyzed hind legs; 3, limp tail with completely paralyzed hind legs; 4, tetraplegia; 5, moribund.

All mice were housed under specific pathogen-free conditions in the animal research facility of the University of Duesseldorf according to EU directive 2010/63/EU. Animal experimentation was approved by local state authorities (Landesamt fuer Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen). Immunization protocols were choosen to prevent that animals became severely sick or died at any time before experimental endpoints. To minimize stress, pain and damage of mice, all animals were handled and treated according to EU directive 2010/63/EU on the protection of animals used for scientific purposes. Animals were daily monitored and termination criteria were definded to protect animals from pain and stress. All animals were sacrificed by cervical dislocation.
To induce active EAE, female C57BL/6 mice (8 weeks, Janvier, Le Genest-Saint-Isle, France) received subcutaneous injections of 200 μg of MOG_35–55 (Biotrend, Cologne, Germany) in complete Freund's adjuvant (CFA; BD Biosciences, Heidelberg, Germany), supplemented with Mycobacterium tuberculosis H37RA (5 mg/ml) (BD) and 500 ng pertussis toxin (Merck Millipore, Darmstadt, Germany) on d0 and d2. The following EAE score was applied [22 (link)]: 0 = no clinical signs; 1 = tail paralysis; 2 = hind limb paresis; 3 = hind limb paralysis; 4 = fore limp paresis; 5 = moribund.

Active immunization was performed on 10 weeks-old male and female mice, IL-20RB^–/–, IL-20^+/–, wild-type, and C57BL/6 with myelin oligodendrocyte glycoprotein (MOG) following standard protocols (Cruz-Orengo et al., 2014 (link)). Briefly, naïve C57BL/6 mice were immunized subcutaneously (s.q.) with 50 μg msMOGp35-55 (GenScript, Piscataway, NJ, United States) and 500 μg Mycobacterium tuberculosis (Mtb) H37Ra peptide emulsified in Freund’s adjuvant (a.k.a. complete Freund’s adjuvant, CFA, both reagents from Difco Laboratories, Detroit, MI, United States). Mice were injected, intraperitoneally (i.p.), with 300 ng pertussis toxin (List Biological Laboratories, Campbell, CA, United States) at the time of immunization and 2 days after. Sham immunizations for control mice were performed by s.q. injection of equal amounts of Mtb, within the CFA; and i.p. injection of 0.9% injectable saline.
Starting 7 days post-immunization (dpi), immunized mice were monitored for clinical manifestations of EAE by following their body weight and graded for disease progression with the following score system: (1) tail flaccidity; (2) hindlimb paresis; (3) hindlimb paralysis; (4) forelimb paresis; and (5) moribund or dead. Mice were monitored until 28 dpi at which point were humanely euthanized.