Evaluating T cell proliferation in immunized pigs

To evaluate the proliferation of T lymphocytes, whole blood (10 mL) was collected from immunized piglets in each group at 42 dpi, and peripheral blood lymphocytes were isolated using a pig peripheral blood lymphocyte isolation kit (TBDsciences, Tianjin, China) in accordance with the manufacturer’s protocol. The lymphocyte proliferation assay was performed as described in our previous study [14 (link)]. Briefly, lymphocytes (4 × 10⁶ cells/mL) were seeded into a 96-well plate with 100 µL RPMI-1640 containing 20% FBS and stimulated with purified APPV E2 protein (10 µg/mL), concanavalin A (10 µg/mL), and 100 µL RPMI-1640 containing 20% FBS. After 72 h of culture at 37 °C, 10 µL CCK-8 reagent (MedChemExpress, Shanghai, China) was added to each well and incubated at 37 °C for 4 h. Finally, the absorbance was measured at 450 nm wavelength, and the stimulation index (SI) was calculated according to the formula: SI = (OD values of immunized groups − OD values of blank control)/(OD values of negative control − OD values of blank control).