Experimental autoimmune encephalomyelitis (EAE) in mice:
For induction of EAE, 8–10-week-old female mice were immunized with the MOG35–55 peptide (100 μg/mouse, AnaSpec) in complete Freund’s adjuvant (Difco Laboratories). On day 0 and day 2, pertussis toxin (100 ng/mouse, List Biological Labs) was injected intraperitoneally. EAE was also induced with adoptive transfer of MOG-stimulated T cells. For this, C57BL/6 mice were immunized with MOG35–55 peptide. ...
Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides Product #180 - Pertussis Toxin from B. pertussis, Lyophilized in Buffer and Product #181 - Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free).
Cell isolation and culture:
Naïve CD4+ T cells were isolated from splenocytes and lymph node cells with the naive CD4+ T cell isolation kit (Miltenyi Biotec). To obtain CNS APCs, brains from newborn mice were collected, dissociated with scissors and digested with collagenase type 4 (2 mg/ml, Worthington Biochemical) for 1 hour at 37 °C. Mononuclear cells in the interphase between 30% and 70% of Percoll (GE healthcare) were isolated by density-cut centrifugation as described38. These cells were cultured for 4 days and then activated by TNF-α (10 ng/ml, BioLegend) and IL-17 (25 ng/ml, BioLegend) in the presence and absence of SCFAs (C2 at 10 mM, C3 at 1 mM, C4 at 0.5 mM) for additional 3 days in DMEM medium (10% FBS). Adherent glial cells, largely composed of microglial cells, were co-cultured as APCs with naïve CD4+ T cells at 1:10 ratio for 5–6 days in the presence of Staphylococcal enterotoxin B (SEB, 5 μg/ml, List Labs) to stimulate naïve CD4+ T cells isolated from the spleen and lymph nodes of unimmunized C57BL/6 mice.