Peripheral blood lymphocyte separation kit

Single-cell suspensions from blood were prepared using a peripheral-blood lymphocyte separation kit (Solarbio, Beijing, China), and flow cytometry was performed to detect the percentages of CD3⁺CD4⁺, CD3⁺CD8⁺ T-cells [57 (link)]. The isolated cells (1 × 10⁶) were incubated with FITC-conjugated anti-chicken CD3 and PE-conjugated anti-chicken CD4 or CD8 (SouthernBiotech) monoclonal antibodies, respectively. The samples were quantified by a flow cytometer (BD LSRFortessa™, USA). Experiments were performed in triplicates and the data were analyzed using FlowJo (Version 7.6.2, Becton, Dickinson and Company, USA).

The peripheral blood lymphocyte proliferation assay was performed using a modified CCK-8 method as described previously^{29 (link)}. Briefly, peripheral blood T lymphocytes were isolated using a Peripheral Blood Lymphocyte Separation Kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. After cell counting, 80 μL of diluted cells in RPMI 1640 medium (Gibco) was seeded into a 96-well plate. To specifically stimulate the proliferation of T lymphocytes, 20 µL of CHN-YC virus (10^6.25 TCID₅₀/100 µL) or purified recombinant truncated E protein (20 mg/mL) was added; the unstimulated control was administered an equal volume of PBS. After 36 h of cell culture at 37 °C, cell proliferation was detected using a Cell Counting Kit-8 (MCE, Shanghai, China) according to the manufacturer’s instructions. The stimulation index calculated from the OD value of each stimulated group, and the OD of mock group is normalized to 1.
The levels of Th1-type cytokine IFN-γ and the Th2-type cytokine IL-4 in serum were measured using duck IFN-γ and IL-4 sandwich ELISA kits (ML Bio, Shanghai, China) following the manufacturer’s instructions.

The peripheral blood lymphocyte proliferation assay (21 (link)) was performed using a modified CCK8 method. Briefly, T-lymphocytes were isolated using a Peripheral Blood Lymphocyte Separation Kit (Solarbio, Beijing, China) according to manufacturer’s instruction. After cell counting, 80 μL diluted cells in RPMI 1640 medium (Gibco) were seeded into a 96-well plate. To specifically stimulate the proliferation of T-lymphocytes, 20 ul CHN-YC virus (10^6.25 TCID₅₀/100 ul) or purified recombinant truncated E protein (20 mg/mL) were added; for mock group, equal volume of PBS was added. After 36 h cell cultured in 37 °C, cell proliferation was detected using a Cell Counting Kit-8 (MCE, Shanghai, China) per manufacturer’s instruction.
The expression of IFN-γ and IL-4 measured by ELISA. Th1-type cytokine IFN-γ and Th2-type cytokine IL-4 in serum at 14 dpi were measured using commercial duck IFN-γ and IL-4 sandwich ELISA kits (mlbio, shanghai, China) following the manufacturer’s instruction.