Cytokine Production Assay in Immune Cells

Splenocytes or peripheral blood mononuclear cells (PBMC) were stimulated with peptide at 2 µg/mL; ionomycin and phorbol myristate acetate (PMA) at 2.0 mg/mL and 0.5 mg/mL, respectively, as positive assay controls; tissue culture medium with 1% DMSO was used as a negative control and processed as previously described [17 (link)]. The following mAb reagents were used: anti-CD107a phycoerythrin (PE)-conjugated mAb, anti-CD3 PerCP-eFluor710, anti-CD8a eFluor 450, anti-IFN-γ PE-Cy7, anti-IL-2 APC, and anti-tumor necrosis factor (TNF)-α fluorescein isothiocyanate (FITC) (all from eBioscience, San Diego, CA, USA) and anti-CD4 allophycocyanin (APC)/Cy7 (BioLegend, San Diego, CA, USA). Fixed cells were acquired on an LSRII flow cytometer (Becton Dickinson, Wokingham, UK). All mice were assayed in triplicates.