Intracellular cytokine staining followed by flow cytometry analysis to determine the number of primary and memory E7-specific CD8+ T cells in tumor-bearing mice treated with apigenin and/or E7-HSP70 DNA vaccine. Groups of C57BL/6 mice (5 per group) were challenged subcutaneously with 1 × 104/mouse of TC-1 tumor cells. Mice were treated with apigenin alone, E7-HSP70 DNA vaccine alone, the combination of apigenin and the E7-HSP70 DNA vaccine. Apigenin was administered intraperitoneally at the dose of 25 mg/kg after TC-1 inoculation and continued for 10 days. Mice were vaccinated with 2 μg/mouse of E7-HSP70 via gene gun, 3 days before TC-1 inoculation and receive a booster dose 7 days after the first vaccination. Untreated tumor challenged mice were used as negative controls. 14 days (for primary immune response) and 42 days (for memory immune response) after tumor challenge, splenocytes from mice were harvested and stained for CD8 and intracellular IFN-γ and then characterized for E7-specific CD8+ T cells using intracellular IFN-γ staining followed by flow cytometry analysis. A & B. Representative data of intracellular cytokine stain followed by flow cytometry analysis showing the number of E7-specific IFNγ+ CD8+ T cells in mice treated with apigenin and/or DNA vaccine at (A) 14 days or (B) 42 days after tumor challenge. C & D. Bar graph depicting the numbers of E7-specific IFN-γ-secreting CD8+ T cells per 3 × 105 pooled splenocytes at (C) 14 days or (D) 42 days after tumor challenge. Data shown are representative of two experiments performed (mean ± SD).