EAE induction and isolation of infiltrated lymphocytes

Experimental autoimmune encephalomyelitis was induced and characterized as previously reported (31). Six- to 8-week-old C57/BL6 mice were immunized with MOG_35-55 peptide (200 μg per mouse emulsified with CFA). Mice were also given pertussis toxin (200 ng per mouse) on day 0 and day 2 via tail vein injection. All mice were weighed and examined daily for clinical symptoms and assigned scores on a scale of 0–5 as follows: 0, no overt signs of disease; 1, limp tail; 2, limp tail and partial hindlimb paralysis; 3, complete hindlimb paralysis; 4, complete hindlimb and partial forelimb paralysis; 5, moribund state or death.

Mice were euthanized during the peak of disease. Following extensive transcardial perfusion with PBS, spinal cords and brains were removed from mice (tissues from 3 mice were pooled for each experiment) and pooled for mononuclear cell isolation. Briefly, tissues were incubated with collagenase D (300 μg/ml) and DNase I (20 μg/ml) in HBSS. After 45 min at 37 degrees, tissues were mechanically dissociated through a 40-μm strainer and washed with PBS. The resultant pellet was fractionated on a discontinuous percoll gradient. Infiltrating mononuclear cells were harvested from the interface, washed, counted, and cultured for 4 h in RPMI containing 10% FCS with PMA (10 ng/ml) and ionomycin (500 ng/ml) in presence of brefedlin A (10 μg/ml) for flow cytometry.