Intracellular cytokine staining followed by flow cytometry analysis to determine the memory recall response of 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, or the combination of apigenin and the E7-HSP70 DNA vaccine as described in Figure 3. Untreated tumor challenged mice were used as negative controls. Sixty days after the last vaccination, the mice were vaccinated twice with E7-HSP70 DNA vaccine to generate the memory recall response. Ten days after the recall vaccination, splenocytes 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) Representative data of intracellular cytokine stain followed by flow cytometry analysis showing the memory recall response of E7-specific IFNγ+ CD8+ T cells in mice treated with apigenin and/or DNA vaccine. (B) Bar graph depicting the numbers of E7-specific IFN-γ-secreting CD8+ T cells per 3 × 105 pooled splenocytes.