Intracellular cytokine staining and flow cytometry analysis

C57BL/6 mice were immunized twice by a subcutaneous injection of rOVA (30 μg), rlipo-OVA (30 μg) or PBS at one-week intervals. Seven days after the final immunization, splenocyte CD8⁺ T cells producing IFN-γ were determined by intracellular cytokine flow cytometry. The peptides used to stimulate the cells were added at a concentration of 2 μg/ml for 18 h at 37°C; then, 1 μl/ml of Brefeldin A (eBioscience), 1 μg/ml ionomycin (Sigma-Aldrich) and 10 μg/ml phorbol 12-myristate 12-acetate (PMA) were added for an additional 4 h before harvesting the cells from the culture. The cells were subjected to intracellular cytokine (IFN-γ) staining using the Intracellular Fixation & Permeabilization Buffer Set (eBioscience) according to the manufacturer's instructions. The cells were washed once with FACS buffer (PBS, 2% FBS and 0.05% sodium azide) and stained with the following monoclonal antibodies: CD16/CD32 (clone 93, eBioscience), CD3ε-PE (clone 145-2c11, Biolegend), CD8a-APC (clone 53–6.7, eBioscience), IFN-γ-PE-Cy7 (clone XMG1.2, eBioscience) and the isotype control antibody (rat IgG1k, eBioscience). Sample acquisition was analyzed using the FACSCalibur (BD Biosciences).