Mog35 55

To determine whether myelin specific T cells require antigen-specific APCs for recruitment to the CNS, EAE was actively induced using a combination of two antigenic peptides in 8–12 week old female recipient mice. 3×10⁶ naïve 2D2-CFP and 3×10⁶ naïve OTII-GFP CD4⁺ T cells were isolated separately by negative depletion using Dynal beads and co-injected into naive recipient mice. 24 hours later, an emulsion of 200 μg MOG35-55 only, 10 μg OVA323-339 only, or 200 μg MOG35-55 and 10 μg OVA323-339 combined (Anaspec, San Jose, CA, USA) with CFA (8 mg/mL H37RA and IFA; Difco Laboratories, Detroit, MI, USA) was injected s.c. bilaterally on the lower back of the recipient mice. Control mice were injected s.c. bilaterally with an emulsion of PBS and CFA. 100 ng PTx (List Biological Laboratories, Inc, Campbell, CA, USA) was administered i.p. on days 0, 1 and 2 to induce EAE.

To determine IFN-γ and IL-17 secretion, cells isolated from CNS draining lymph nodes were adjusted to 5 × 10⁶ cells (c)/ml in RPMI 1640 medium, containing 8.6% FCS, 4 mM glutamine, 1 mM sodium pyruvate, 0.1 mM MEM non-essential amino acids, 200 μg/ml penicillin, 200 μg/ml streptomycin, 25 μg/ml gentamicin, and 50 μM 2-ME and stimulated with MOG_35–55 (MEVGWYRSPFSRVVHLYRNGK) (Ana Spec, Fremont, CA) in Costar® 96-well round-bottom culture plates (Thermo Fisher Scientific Inc., Waltham, MA) for 96 h. Cell-free supernatants were collected and cytokines were quantified by ELISA using Ready-SET-Go!® ELISA kits (eBioscience, San Diego, CA). To determine TNF, TNFR1, and TNFR2 concentrations, serum samples were collected and quantified using eBioscience Ready-SET-Go!^® ELISA kits (TNF and TNFR2) or the mouse TNFR1 DuoSet^® ELISA kit (R&D Systems, Minneapolis, MN). All cell-free culture supernatants and serum samples were stored at −80°C until use. The absorbance (450 nm) minus background (570 nm) of the colorimetric reactions were quantified using a Synergy^™ 4 spectrophotometry microplate reader (BioTek, Winooski, VT). The lower limit of sensitivity of the TNF assay was 8 pg/ml. The recognition of TNF by the Ready-SET-Go!^® assay is not affected by 1000-fold excess soluble TNFR1 or TNFR2.