Multi-color ICS was performed as previously reported (29 (link)). Briefly, 1 × 106 freshly isolated mouse splenocytes were incubated with peptide pool s for 2 h. Brefeldin A (eBioscience) was added to block cytokine secretion for 8 h. Cells were stained with antibody cocktails (anti-CD3-PerCP, anti-CD4-APC and anti-CD8-FITC) (BD Bioscience) for 30 min at room temperature, permeabilized using Cytofix/Cytoperm buffer (BD Bioscience), and stained with anti-IFN-γ-PE, anti-TNF-α-PE-Cy7, and anti-IL-2-APC-Cy7 (BD Bioscience). Samples were subjected to flow cytometric analysis using the FACSArial instrument and data were processed with FlowJo 7.6 software (Tree Star, Inc.).
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