Multi-color Flow Cytometry Analysis

All antibodies were purchased from BD Biosciences (San Jose, CA) or eBioscience, Inc. (San Diego, CA) as published (Offner et al 2006b (link), Offner et al 2006c (link)). Four-color (FITC, PE, APC and PI/PerCP/PECy7) fluorescence flow cytometry analyses were performed to determine the phenotypes of splenocytes and brain leukocytes as previously published (Offner et al 2006a (link)). Single-cell suspensions were washed with staining medium (PBS containing 0.1% NaN₃ and 0.5% bovine serum albumin, Sigma, Illinois) and incubated with combinations of the following monoclonal antibodies: CD4 (GK1.5, BD Pharmingen), CD8a (53–6.7, BD Pharmingen), CD11b (M1/70, eBioscience), CD45 (Ly-5, BD Pharmingen), CD19 (1D3, BD Pharmingen), CD1d (1B1, BD Pharmingen), CD122 (TM-β1 BD Pharmingen), MHCII (2G9, BD Pharmingen), CD69 (H1.2F3, BD Pharmingen) for 10 min at 4°C. One mL of staining buffer was added to wash the cells. Propidium iodide (PI) was added to identify dead cells whenever only 3 channels on the FACSCalibur were used for detection of fluorescent antibody staining. The FoxP3 staining kit was used according to the manufacturer's protocol (eBioscience) as previously described (Zhang et al 2010 (link)). FACS data acquisition was performed on FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and data were analyzed using FCS express software (De Novo Software, Los Angeles, CA).