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).