Tnfα bv510

Splenocytes were isolated from the vaccinated mice and seeded into 24‐well plates to quantify the percentages of antigen‐specific T cells; ≈10⁶ cells were added per well. Subsequently, stimulated the cells with mosaic peptides pool (GenScript) at a final concentration of 2 g mL⁻¹ and co‐stimulated with 2 µg mL⁻¹ anti‐CD28 (1000× Biolegend 102116) at 37 °C with 5% CO₂ for 1 h. DMSO was used as a negative control, and PMA/ionomycin was used as a positive control. Splenocytes were then blocked with 5 µg mL⁻¹ brefeldin A (TargetMOL T6062/20350156) and 2 µM monensin (TargetMOL T1033/22373780) for 5 h to prevent cytokines from passing through the cell membrane. Then the cells were extracellularly stained with fluorochrome‐conjugated monoclonal antibodies for 20 min within PBS containing 0.5% BSA on ice. The following antibodies were used: FVD (Invitrogen 65‐0865‐14), CD3‐ PerCP‐Cy5.5 (Biolegend 100328), CD4‐PE‐Cy7 (Biolegend 566939), CD44‐FITC (Biolegend 103021), followed by treatment of 200 µL IC Fixation buffer (BD Biosciences) overnight at 4 °C. The next day, cells were performed with the 1 mL Permeabilization buffer (Invitrogen 00‐8333‐56). Then the antibodies IL‐2‐BV421(Biolegend 503820), TNF‐α‐BV510 (Biolegend 506339), IFN‐γ‐APC (Biolegend 505810), IL‐4‐PE (Biolegend 504104) were used for the intracellular staining and the flow cytometry analysis.