Bordetella pertussis toxin

For adoptive transfer EAE, age- and sex-matched donor mice were subcutaneously immunized with 150 µg MOG_35–55 peptide (Genemed Synthesis, Inc.) emulsified in complete Freund’s adjuvant (5 mg/ml heat-killed M. tuberculosis), followed by an intraperitoneal (IP) injection of 250 ng of Bordetella pertussis toxin (List Biological Laboratories, Inc.) in 200 µl of PBS at 0 and 2 days postimmunization. Ten days postimmunization, spleens and lymph nodes were collected and mechanically disrupted to generate a single-cell suspension. For TH17-EAE, the cells were cultured at 2.5 × 10⁶ cells/ml for 72 h and stimulated with 10 µg/ml MOG_35–55, 10 ng/ml IL-23, and 10 µg/ml IFN-γ antibody in complete RPMI media (23 (link)). For TFH-EAE, cells were cultured with 10 µg/ml MOG_35–55, 20 ng/ml IL-6, 20 ng/ml IL-21, 10 µg/ml IFN-γ antibody, 10 µg/ml IL-4 antibody, and 20 µg/ml TGF-β antibody in complete RPMI media as previously described (24 (link)). On Day 3, cells were collected and 5 × 10⁶ cultured cells were transferred into healthy recipient mice by IP injection.
Mice were monitored daily for clinical signs. Paralysis was assessed using a standard clinical score ranging from 0 to 5 with scores corresponding to the following phenotypes: 0, no disease; 1, loss of tail tone; 2, partial hind-limb paralysis; 3, complete hind-limb paralysis; 4, forelimb paralysis; and 5, moribund/dead.