Multi-parameter Flow Cytometry Analysis

A CNS and spleen leukocyte suspension was obtained as described previously (Carrillo‐Salinas et al., 2017 (link)). Isolated cells were incubated with anti‐CD16/CD32 (Affymetrix Inc.) for FcR blockade and labeled with anti‐mouse antibodies: PE‐conjugated anti‐CD44 (2.4 μg/ml), PE‐conjugated anti‐CD274 (2.4 μg/ml), PerCP‐Cy5.5‐conjugated anti‐CD4 (1.2 μg/ml), PECy7‐conjugated anti‐Ly6c (1.2 μg/ml), APC‐Cy7‐conjugated anti‐CD11b (1.2 μg/ml), APC‐conjugated anti‐CD62L (2.4 μg/ml), APC‐conjugated anti‐CD25 (2.5 μg/ml), and APC‐conjugated anti‐CD1d (2.4 μg/ml; all from eBioscience); APC‐conjugated anti‐P2yR12 (2.5 μg/ml) and APC‐Cy7‐conjugated anti‐CD3 (1.2 μg/ml) (both from Biolegend); PE‐conjugated anti‐CD5 (2.2 μg/ml), PerCP‐Cy5.5‐conjugated anti‐B220 (2.4 μg/ml), PerCP‐Cy5.5‐conjugated anti‐CD45 (1.2 μg/ml), PECy7‐conjugated anti‐CD8 (1.2 μg/ml) and PECy7‐conjugated anti‐CD19 (2.4 μg/ml; all from BD Pharmingen). The cells were fixed for 30 min with fixation buffer (Affymetrix Inc.). For FoxP3 detection, the cells were suspended in Fixation/Permeabilization buffer for 30 mins prior to staining with an anti‐FoxP3 antibody (3 μg/ml; BD Pharmingen). At least 50,000 events were registered in each experiment on a FACSAria flow cytometer (BD Biosciences), excluding duplets from the analysis. The data were analyzed using FACSDiva analysis software (BD Biosciences).