Multiparameter Flow Cytometry of Immune Cells

Cells isolated from spleen, lymph nodes, brain, and spinal cord were analysed using a FACS Canto II flow cytometer (BD, Franklin Lakes, NJ) as described^{14 (link)}. The following antibodies were used for phenotypic analysis: mouse anti-NK1.1 IgG2a, hamster anti-CD11c IgG, rat anti-Ly6C IgG2c, rat anti-Ly6G IgG2a, mouse anti-FoxP3 IgG1, mouse anti-CX3CR1 IgG2a, and mouse anti-CD45.1 IgG2a from Biolegend (San Diego, CA) and rat anti-CD4 IgG2a, rat anti-CD8α IgG2a, rat anti-CD25 IgG1, rat anti-I-A^b (MHC-II) IgG2a, rat anti-CD11b IgG2b, mouse anti-CD45.2 IgG2 and rat anti-Gr1 IgG2b from BD Biosciences (San Jose, CA) and rat anti-PD-L1 IgG2a and rat anti-F4/80 IgG2a from eBiosciences (San Diego, CA). Fcγ receptors were blocked by the addition of purified anti-CD16/32 antibodies (Fc Block; 1 μg/10⁶ cells; BD Biosciences) 15 minutes prior to surface staining. For intracellular cytokine staining, GolgiStop (1 μg/10⁶ cells; BD Biosciences), ionomycin (500 ng/ml; Sigma) and PMA (50 ng/ml) were added to the Con A-stimulated splenocyte cultures 5 hours prior to surface antibody staining. After extracellular staining, cells were fixed and permeabilized using Transcription Factor Buffer (BD Biosciences, USA) and stained with rat anti-IFN-γ IgG1 (BD Biosciences) following the manufacturer’s instructions and analyzed using BD FACS Canto II flow cytometer (BD Biosciences).