Cytotoxicity assay in vitro

Peripheral blood mononuclear cells (PBMCs) were isolated from pig whole blood using density gradient centrifugation with Histopaque (Sigma-Aldrich). Briefly, whole blood was diluted at the ratio of 1:1 with PBS and carefully layered over 6 ml of Histopaque solution. Tubes were centrifuged at 400 × g for 30 min at room temperature. PBMCs were then carefully collected from the buffy coat layer, washed twice with PBS, and frozen at −80°C until ready to use. PBMCs were resuscitated and cultured for 3 days in RPMI 1640 GlutaMAX medium (Thermo Fisher Scientific), 10% FBS, and 1% penicillin-streptomycin (Thermo Fisher Scientific). PBMCs were either maintained in medium alone or activated with phytohemagglutinin (PHA) (Sigma-Aldrich) (5 μg/ml) for 3 days. PICs (P6) were plated at 1 × 10⁵ per well in a 24-well plate, 1 day before co-culture. PBMCs were harvested and co-cultured with PICs in RPMI medium as follows; no PBMCS, 1:10, 1:20, and 1:40. After 4 h of co-culture, PBMCs were discarded; PICs were harvested and subsequently labelled with anti-CD45 antibody/Annexin V-PE Apoptosis Detection Kit I (BD Biosciences) and analyzed by flow cytometry using a FACSCanto II and FACS DIVA V8.0.1 software (BD Biosciences).