Macrophage cultures and transfer to EAE mice
Immunized C57BL/6 mice were sacrificed and bone marrow cells from femoral and tibial bones were collected in RPMI-1640 complete medium(Gibco, Waltham, MA) on days 2, 5, and 8 p.i. After red blood cell lysis, cells from the bone marrow were cultured in RPMI-1640 complete medium supplemented with penicillin and streptomycin(1%; Hyclone, Logan, UT), fetal bovine serum(10%; Sigma Aldrich, St Louis,MO), IL-4(10ng/ml; Pepro-Tech, Rocky Hill, NJ), and macrophage colony-stimulating factor(M-CSF) (10ng/ml; Pepro-Tech, Rocky Hill, NJ). On days 3, 5, and 7 post-culture, half of the previous medium was replaced with fresh medium and the concentrations of IL-4 and M-CSF were increased to 20ng/ml. M2 macrophages were harvested on day 8 post-culture using ethylene diamine tetra acetic acid(EDTA; Sigma) and incubated with MOG35–55(20µg/ml) peptide for 4h at 37°C. Then the cell concentration was set to 10×106/ml, and the cells were transferred to EAE mice(0.1ml, 1×106 cells/mouse) on days 10, 13 and 16 p.i. via the caudal vein. And the control group were treated with PBS. Experiments were replicated three times(n = 10).