Mixed lymphocyte reaction (MLR)

To evaluate the adjuvanticity of B. bronchiseptica , the cultured DCs were treated with LPS, B. bronchiseptica for 2 days, and then co-cultured with allogeneic spleen cells. Allogeneic spleen cells were harvested from Balb/c mice. The treated DCs were incubated with 50 µg/ml mitomycin C for 30 min before the co-culture setup. The DCs and allogeneic spleen cells were cultured in 96-well plates at a concentration of 1 × 10⁵ cells/ml and 2 × 10⁶ cells/ml, respectively. As a control, only allogeneic spleen cells were cultured. After 5 days, 10 µl/well of CCK-8 solution was added and incubated at 37℃ for 4 h. The optical density was measured at 450 nm with a microplate reader.