Intracellular cytokine staining with flow cytometry analysis to determine the proportion of specific IFN-γ -expressing CD8+ T cells. Splenocytes from mice (n = 3 per group) immunised with virus rVVJ16/18E7E6 and virus rVVJ1175 were cultured and stimulated with either HPV16E749-57 or HPV18E667-75 peptide. Splenocytes without peptide stimulation were used as a negative control. The splenocytes were stained for both CD8+ and intracellular IFN-γ (A) Representative intracellular cytokine staining. The number of CD8+ IFN-γ + T cells in 1 × 106 splenocytes are indicated in the upper right corner. (B) Bar graph depicting the number of specific IFN-γ expressing CD8+ T cells per 1 × 106 splenocytes (mean ± SD) following in vitro stimulation in two independent experiments. Asterisks represent statistically significant differences relative to the rVVJ1175 virus control (* p < 0.05, ** p < 0.001).