Methyl thiazolyl tetrazolium (mtt)

The lymphocyte proliferation assay was performed as previously described [32 (link)], but with some modifications. Briefly, 1 mL of blood collected from the wing vein and twice diluted with PBS was overlain on the same volume of lymphocyte separation medium (1.077 specific gravity; Sigma-Aldrich) and centrifuged at 500 × g for 20 min. The peripheral lymphocytes were harvested and checked by staining with trypan blue dye. Viable lymphocytes suspension (5 × 10⁵ cell/well) were plated into 96-well tissue culture plates in triplicate and incubated in 100 µL complete RPMI-1640 medium with or without NDV (10⁴ EID₅₀/well) and with 10 µL/well conA (0.5 mg/mL; Tiangen, China,) as a positive control. After 72 h, 10 µL/well MTT (5 mg/mL; Tiangen) were added to each well and the plates were incubated for 4 h at 37℃. Then, 150 µL per well of dimethyl sulfoxide was added and the plates were further incubated for 10 min at 37℃. OD was measured with an ELISA reader (Perlon, China) at a wavelength of 570 nm. All samples were analyzed in triplicate. The stimulation index (SI) was calculated according to the formula: SI = mean OD of stimulated cells/mean OD of un-stimulated cells [32 (link)].