Ficoll hypaque density gradient

A 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium-bromide (MTT) assay was used to evaluate T lymphocyte proliferation. Briefly, lymphocytes were isolated from heparinized swine blood collected 35 dpi by Ficoll-Hypaque density gradient centrifugation for 20 min at 5,000 × g at 4℃, washed three times with modified Hanks Balanced Salt Mixture (HBSS; Gibco, USA), and seeded at 5 × 10⁵ cells/well into 96-well U-bottom culture plates (Nunc, Denmark) in complete Roswell Park Memorial Institute (RPMI) medium 1640 (Gibco) with 10% FBS. Each sample was plated and assayed in triplicate. The T lymphocytes were stimulated with the field isolate PRRSV Guizhou strain at a multiplicity of infection (MOI) of 1 as the specific antigen. Cell lysate extracted from mock-infected Marc-145 cells with PBS was used as a negative control, and concanavalin A (Con A, 5 µg/mL; Sigma, USA) served as a positive control. After incubation at 37℃ for 45 h in 5% CO₂ and subsequent incubation for 4 h with MTT of 0.05% concentration (Sigma), proliferation was detected as previously described [30 (link)]. The plates were read at 570 nm with a microplate reader (Thermo Fisher Scientific). The stimulation index (SI) was calculated as the ratio of the average optical density (OD) of wells containing antigen-stimulated cells to the average OD of wells containing cells cultured with medium alone.