Apc anti mouse perforin

Spleens were isolated from immunized C57BL/6 mice 3 days after the last DC administration (n=3). Prepared splenocytes (1×10⁶ cells/well) were restimulated in 24-well plates with 5 µg/mL freshly-prepared vaccine protein for 6 h in the presence of recombinant mouse IL-2 (20ng/ml; PeproTech). 50ng/mL PMA, 1ug/ml Ionomycin and 10 µg/mL Brefeldin A (Absin) was added to accumulate intracellular cytokines. After restimulation, the cells were firstly incubated with anti-mouse CD3, CD4 and CD8 antibodies for surface staining. Subsequently, intracellular staining for IL-2, IFN-γ, Granzyme B and Perforin were performed after these cells were fixed and permeabilized. Fixable viability dye was used to gate out dead cells. Data were collected on FACS verse (BD Biosciences) and analyzed with Flow Jo software. The antibodies used in this study were including FITC anti-mouse CD3 (Thermo Fisher), PE anti-mouse CD4 (BD Biosciences), PerCP-Cy™5.5 anti-mouse CD8a (BD Biosciences), PE-Cy7 anti-mouse IL-2 (BD Biosciences); BV510 anti-mouse IFN-γ (Biolegend); BV421 anti-mouse Granzyme B (Biolegend); APC anti-mouse Perforin (Biolegend); Fixable Viability Stain 780 (BD Biosciences); Fixation/Permeabilization Kit (BD Biosciences).