ELISPOT Assays

Lymphocytes were isolated from the brain by separating the hemispheres and homogenizing the tissue through a 70 micron screen. The homogenate was spun over a Ficoll®-Paque gradient to separate the lymphocytes from brain tissue. Lymphocytes (1×10⁵ cells/well) were cultured in media alone or media supplemented with antigen or the mitogen concanavalin A (ConA; Sigma) for 48 hours in 96 well plates (Multiscreen®-IP, Millipore). ELISPOT assays were used to detect rat MBP (NeoBioSci™) and ovalbumin (OVA; Sigma) specific secretion of interferon (IFN)-γ, interleukin (IL)-17 and transforming growth factor (TGF)-β1. Antigens were used at a concentration of 50 μg/mL and ConA at 5 ug/mL. Responses were assessed in triplicate.

Plates were developed using standard protocols (R & D Systems). Spots were counted with the aid of a semi-automated system (AID iSPOT®) and expressed as the ratio of the relative increase in antigen-specific IFN-γ secreting cells to that of TGF-β1 secreting cells (TH1 response) or as the ratio of the relative increase in antigen-specific IL-17 secreting cells to that of TGF-β1 secreting cells (TH17 response).