Bordetella pertussis toxin

Fourteen-week-old mice were immunized subcutaneously with 150 μg of myelin oligodendrocyte glycoprotein peptide (residues 35–55; MOG_35–55; Bio-Synthesis, TX) emulsified in complete Freund’s adjuvant (CFA; Chondrex, Redmond, WA) containing 2.5 mg/ml heat-killed Mycobacterium tuberculosis. Mice were injected intraperitoneally with 250 ng Bordetella pertussis toxin (Chondrex) the day of, and again, 2 days following MOG_35–55 immunization. At 21–28 days post-immunization, mice were euthanized for histological analyses. The mice were euthanized by CO₂ asphyxiation for 10 min from a compressed gas tank followed by decapitation or cardiac perfusion. Following perfusion, mice were immediately decapitated. EAE clinical manifestations of progressive ascending paralysis were assessed daily using a standard scoring system: 0 = no overt symptoms, 1= loss of tail tone, 2 = hind limb paresis, 3 = complete hind limb paralysis, 4 = hind limb paralysis and forelimb paresis, and 5 = moribund or dead. The mice were weighed daily to ensure weight loss did not exceed 25% of starting weight.

Eight- to nine-week-old mice were immunized subcutaneously with 150 μg myelin oligodendrocyte glycoprotein (MOG_35–55; Bio-Synthesis, City, TX) emulsified in complete Freund’s adjuvant (CFA; Difco Laboratories, Detroit, MI) containing 2.5 mg/ml heat-killed Myobacterium tuberculosis. Mice were injected intraperitoneally with 250 ng Bordetella pertussis toxin (Chondrex, Redmond, WA) the day of, and 2 days following, MOG_35–55 immunization. At 21 days post-immunization, the mice were euthanized for the histological analyses. EAE clinical manifestations of progressive ascending paralysis were assessed daily using a standard scoring system: 1 = loss of tail tone, 2 = hind limb paresis, 3 = complete hind limb paralysis, 4 = hind limb paralysis and forelimb paresis, and 5 = moribund or dead. Mice were weighed daily to ensure weight loss did not exceed 25% of starting weight. All EAE data were gathered in two independent experiments from cohorts of the same 12 WT mice and ten KO mice.