Isotonic percoll

Mice were perfused with PBS before spleens, lymph nodes, and CNS were harvested; the meninges were removed from brains, and cells were isolated from all tissues by cell straining (70 µm for spleens and lymph nodes, 100 µm for CNS). For spleen cells, erythrocytes were lysed by incubating the cells on ice for 3 min with lysing buffer (BD Pharm Lyse); leukocytes from blood were isolated by using a Ficoll gradient (Cedarlane); CNS homogenates were separated into neuronal and leukocyte populations by discontinuous density gradient centrifugation using isotonic Percoll (Amersham). For surface marker stainings, cells were incubated with fluorochrome-conjugated antibodies at the recommended dilution or with isotype control antibodies listed above for 20 min at 4°C. For intracellular cytokine staining, isolated leukocytes were stimulated with PMA (10 ng/ml)/ionomycin (1 µg/ml; Sigma-Aldrich) in the presence of Brefeldin A (10 µg/ml; Sigma-Aldrich) at 37°C for 6 h. The intracellular staining kit (eBioscience) was used to permeabilize and fix the cells before staining for Foxp3 and intracellular cytokines. Stained cells were analyzed by flow cytometry (FACSCelesta, Becton Dickinson).