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.