Multiparameter Flow Cytometry Analysis of Leukocytes

A CNS or spleen leukocyte suspension was obtained as described previously.^{49 (link)} Freshly isolated cells (10⁶) were incubated with an anti-mouse CD16/CD32 FC (eBioscience) and labeled with anti-mouse antibodies: PE anti-CD25; PE anti-CD3; PerCP-Cy5.5-conjugated anti-CD45; PerCP-Cy5.5-conjugated anti-B220; PE-Cy7-conjugated anti-CD39; PE-Cy7-conjugated anti-CD8a; PE-Cy7-conjugated anti-CD19; APC anti-CD3; APC-Cy7-conjugated anti-CD4; APC-Cy7-conjugated anti-CD11b (all from BD Pharmingen); APC anti-CD3; and PE-Cy7-conjugated anti-CD39 (both from eBioscience). To detect Foxp3, the cells were suspended in the fixation/permeabilization buffer and stained with a PE anti-Foxp3 antibody (BD Pharmingen). Cell viability was assessed with the LIVE/DEAD Fixable Green Dead Cell Stain Kit or LIVE/DEAD Fixable Red Dead Cell Stain Kit (Life Technologies Corporation). At least 10,000 events were acquired in each experiment on a FACSaria flow cytometer (BD Biosciences) and the data were analyzed using FlowJo software (v.10; Tree Star).