Intracellular IFN-γ staining and flow cytometry

Splenocytes from the immunized mice were treated with ammonium chloride-tris (ACT) buffer for 3 min at room temperature to remove red blood cells. The cells were then washed twice with RPMI 1640 medium and resuspended in RPMI/10FBS at a concentration of 1 × 10⁷ cells/ml. The cells (2 × 10⁶ cells) were incubated for 4 h at 37 °C in the presence or absence of peptide at a concentration of 10 µg/ml with Golgistop stock solution (monensin solution; BD Biosciences, San Jose, CA) diluted 1:1,500. The cells were then washed twice with 2% bovine serum albumin (BSA) in phosphate buffered saline (PBS) followed by staining with fluorescein isothiocyanate (FITC)-conjugated anti-CD8 (clone KT15; MBL, Nagoya, Japan) and phycoerythrin-indotricarbocyanine (PE/Cy7)-conjugated anti-CD4 (clone GK1.5; BioLegend, San Diego, CA) monoclonal antibodies (mAbs) for 30 min at 4 °C. The cells were washed twice and then intracellular cytokine staining (ICS) was performed by using a BD Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s protocol. ICS for IFN-γ was performed with PE-conjugated anti-IFN-γ (clone XMG1.2; BD Biosciences) mAbs for 30 min at 4 °C. The cells were washed twice, resuspended in PBS with 2% BSA, and then analyzed by flow cytometry with a SA3800 spectral analyzer (Sony Corporation, Tokyo, Japan).