2.13. Cytokine production in spleen cell culture
The mouse spleens were grinded and lysed to obtain the splenocytes suspension. Each suspension was resuspended to 5×105cells/mL in RPMI 1640 medium with 10% heat-inactivated FBS, 0.05 mM 2-mercaptoethanol, 100 IU/mL penicillin, and 100 μg/mL streptomycin. The cells were subsequently stimulated with 10 μg/mL of ORF8 and RPMI 1640 medium in 5% (v/v) CO2 at 37 °C for 60 h, respectively. The culture supernatants were collected at 68 h after stimulation to determine the levels of IFN-γ, IFN-β, TNF-α, and IL-5, using the mouse cytokine ELISA kits as instructed by the manufacturer’s protocol. Experiments were performed in triplicate and repeated at least three times independently.