Complete freund s adjuvant incomplete freund s adjuvant ifa

An antibiotic cocktail (ABX) consisting of Ampicillin (1g/L), Metronidazole (1g/L), Neomycin Sulfate (1g/L), and Vancomycin (0.5g/L) dissolved in water was used to deplete the microbiota of mice. Female mice were gavaged with 200ul of ABX once a day for three consecutive days. Mice were immunized after three days of antibiotic oral gavage or sham treatment with 200μg mouse MOG-35-55 peptide (PolyPeptide Laboratories, San Diego, CA) in 200μg Complete Freund’s adjuvant (Incomplete Freund’s adjuvant (IFA, Sigma-Aldrich, St. Louis, MO)) with heat-killed Mycobacterium tuberculosis (Mtb, Difco, Detroit, MI) subcutaneously along the flanks at four sites. Additionally, mice were administered 75ng of pertussis toxin (Ptx, List Biologicals, Campbell, CA) via an intraperitoneal (i.p.) injection on the day of immunization and 200ng i.p. two days later. All mice were monitored daily for weight loss and clinical signs of EAE disease. Mice were scored using the following scale: 0 = normal; 1 = limp tail or mild hind limb weakness; 2 = moderate hind limb weakness or mild ataxia; 3 = moderately severe hind limb weakness; 4 = severe hind limb weakness or mild forelimb weakness or moderate ataxia; 5 = paraplegia with no more than moderate forelimb weakness; 6 = paraplegia with severe forelimb weakness or severe ataxia or moribund condition.

Female uMT^−/− mice were implanted subcutaneously with 2.5mg/60-day release 17β-estradiol pellets (E2, Innovative Research of America, Sarasota, FL) or sham-treated control) one week prior to immunization with 200µg mouse (m) MOG-35-55 peptide (PolyPeptide Laboratories, San Diego, CA) in 200µg Complete Freund’s adjuvant (Incomplete Freund’s adjuvant (IFA, Sigma-Adrich, St. Louis, MO) complemented with heat-killed Mycobacterium tuberculosis (Mtb, Difco, Detroit, MI). Mice received pertussis toxin through intraperitoneal injection (Ptx, List Biologicals, Campbell, CA) on the day of immunization (75ng) and 2 days later (200ng).
All mice were monitored daily for clinical signs of disease and scored using the following scale: 0=normal; 1=limp tail or mild hind limb weakness; 2=moderate hind limb weakness or mild ataxia; 3=moderately severe hind limb weakness; 4=severe hind limb weakness or mild forelimb weakness or moderate ataxia; 5=paraplegia with no more than moderate forelimb weakness; and 6=paraplegia with severe forelimb weakness or severe ataxia or moribund condition. Mice were scored daily and were evaluated for incidence, day of onset, day of maximal clinical signs (peak) and for total disease score over the course of the experiment (Cumulative Disease Index, CDI). Mean ± SEM were calculated for these parameters for each experimental group.