Brains and spinal cords from perfused mice were homogenized separately as previously described [47, 48] . Homogenates were centrifuged at 4506g for 7 min at 4uC. Supernatants were stored at -80uC for cytokine determination (see below). Cells were resuspended in 30% Percoll (Amersham Biosciences, Piscataway, NJ) and isolated by centrifugation (8006g for 30 min at 4uC) onto 70% Percoll cushions. Non-specific binding was inhibited by incubation with anti-CD16/CD32 (2.4G2; BD Biosciences, San Diego, CA) and a 10% mixture of normal goat, human, mouse and rat serum for 10 min on ice. FITC, PE, PerCP, and APC conjugates with monoclonal antibodies (mAb) used to identify and quantify microglia and inflammatory cells were: CD4 (GK1.5), CD8a (53-6.7), CD45 (30-F11), MHC class II (2G9), Ly6G (1A8) (BD Biosciences), and F4/80 (Serotec, Raleigh, NC). Microglia and inflammatory cells were distinguished based on differential CD45 expression. CD4 + T cells were identified as CD45 hi CD4 + , CD8 + T cells as CD45 hi CD8 + , macrophages as CD45 hi F4/80 + and neutrophils as CD45 hi Ly6G + MHC class II -. MOG specific induction of cytokines was determined by stimulation of 1610 6 CNS cells with 20 mg/ml peptide for 6 h at 37uC with GolgiStop (BD Biosciences) added for the last 4 h. Intracellular cytokines were detected with FITC-anti-IFN-c (clone XMG1.2; BD Biosciences) and PE-anti-IL-17 (clone TC11-18H10; BD Biosciences). Intracellular Foxp3 was detected by staining for cell surface markers, followed by permeabilization with Fixation/ Permeabilization Reagent (eBioscience, San Diego, CA) and incubation with PE-labeled anti-Foxp3 (FJK-16s; eBioscience). Cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences) using CellQuest Pro software (BD Biosciences). Data was analyzed using FlowJo (7.6.1) software (Tree Star Inc., Ashland, OR).