Proliferation assays

Female SJL mice at 9 weeks of age were immunized with 100 µg/mouse of PLP_139-151 peptide in 50% CFA in their foot-pads and treated with, or without, homotaurine in their drinking water (0.25 mg/ml). Nine days later, CD4⁺ T cells were purified from their popliteal lymph nodes (the responding T cells) and T cell-depleted splenic mononuclear cells (APCs) were prepared by negative selection using microbeads and magnetic sorting (BD PharMigen). The responding T cells were mixed with APCs in a ratio of 5:1, and were stimulated in triplicate with the indicated concentrations of PLP_139-151 in 1% fetal calf serum HL-1 medium for 3 days. The mixed cells without peptide stimulation served as the negative control. During the last 16-h incubation, individual wells of cells were treated with 1 µCi ³H-thymidine and the T cell proliferation was determined by a β-counter.