In Situ Tolerance within the Central Nervous System as a Mechanism for Preventing Autoimmunity

Brabb, Thea; von Dassow, Peter; Ordonez, Nadia; Schnabel, Bryan; Duke, Blythe; Goverman, Joan

Lymphocyte Isolation. Mice were anesthetized with a mixture of xylazine (7 mg/kg), ketamine (100 mg/kg), and pentobarbital (50 mg/kg) diluted in saline and administered by intraperitoneal injection. Deeply anesthetized mice were perfused with 50 ml of heparinized PBS via the left ventricle of the heart. The spinal cords were removed via insufflation and the brain was dissected. Both tissues were minced, dissociated with a 5-ml syringe plunger, and strained through wire mesh (Small Parts). After centrifugation, cells were resuspended in 37% Percoll (Amersham Pharmacia Biotech), centrifuged at 2,118 g for 15 min, washed, and counted. To prepare cells for proliferation experiments, pooled CNS samples were washed twice with 37% Percoll, resuspended in 30% Percoll, and layered over 70% Percoll. Cells harvested from the gradient interface were washed and resuspended in complete media (DMEM; GIBCO BRL) supplemented with essential amino acids (Irving Scientific), 9.5% FCS, 1.9 mM l -glutamine, 0.95 mM sodium pyruvate, 43 M ␤ -mercaptoethanol, 95 U/ml penicillin G, 95 g/ml streptomycin sulfate, and 0.1 mM nonessential amino acids. The amount of contamination of the CNS mononuclear cell preparation with peripheral cells was assessed using proflavin staining. Proflavin is a nuclear dye that crosses the endothelium of most blood vessels easily but does not cross the blood-brain barrier (18, 19) . In two separate staining experiments in which proflavin hydrochloride (20 g/ml) was administered intravenously, Ͼ 80% of the CNS mononuclear cells did not stain positively for proflavin (data not shown).