For mice immunized with OVA antigen, splenocytes were collected 10 days after the third immunization, and stimulated with antigen (OVA, 50 μg/mL; splenocytes, 4×10 6 cells/mL) for 85 h at 37°C in a humid atmosphere with 5% CO 2 . The supernatant was collected by centrifugation (500×g, 5 min). IFN-γ, IL-4, and granzyme B levels in the supernatant were measured by Ready-to-use Sandwich ELISA kits (eBioscience, San Diego, CA), and IL-5, IL-6, IL-12, and TNF-α levels in the supernatant were detected by ProcartaPlex Mouse Essential Th1/Th2 Cytokine Panel (eBioscience, San Diego, CA), according to the manufacturer's instructions.
For mice immunized with H5N1 influenza split vaccine, splenocytes were collected 14 days after the second immunization, and stimulated with HA (2.5 μg/mL; splenocytes, 5× 10 6 cells/mL) or IYSTVASSL peptide (1 μg/mL; splenocytes, 5×10 6 cells/mL) for 65 h at 37°C in a humid atmosphere with 5% CO 2 . Concentrations of IFN-γ and granzyme B in the culture supernatant were measured by Ready-to-use Sandwich ELISA kits (eBioscience, San Diego, CA).
For mice immunized with H5N1 influenza split vaccine, splenocytes were collected 14 days after the second immunization, and stimulated with HA (2.5 μg/mL; splenocytes, 5× 10 6 cells/mL) or IYSTVASSL peptide (1 μg/mL; splenocytes, 5×10 6 cells/mL) for 65 h at 37°C in a humid atmosphere with 5% CO 2 . Concentrations of IFN-γ and granzyme B in the culture supernatant were measured by Ready-to-use Sandwich ELISA kits (eBioscience, San Diego, CA).