Lymphocyte separation medium

Heparinised peripheral blood (20 ml) was taken from the precaval vein of the vaccinated pigs and control animals at 3, 5, 7, 9, 11, 15 and 18 DPI. Fresh peripheral blood was diluted with an equal amount of phosphate buffered saline (PBS) and was then slowly transferred to a 15-ml glass tube containing lymphocyte separation medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The ratio of diluted peripheral blood to lymphocyte separation medium is 1:1. Following by horizontal gradient centrifugation at 400 × g for 20 min at 20 °C, PBMCs were separated and washed with cold PBS, and were then centrifuged at 450 × g for 10 min at 4 °C.

Tumours were excised and cut into small pieces after removal of blood vessels and connective tissue. To isolate blood cells, tumour tissue was incubated for 1 h, with occasional shaking, in an enzyme mixture that consisted of 1 mg /ml of collagenase D, 20 mg /ml of DNase I (Roche), and 10% fetal calf serum in RPMI-1640 at 37 °C. The digested tissue was filtered through a 70 μm nylon mesh, and the resultant cells were washed twice in PBS. Mononuclear cells were obtained with Lymphocyte Separation Medium (Sigma-Aldrich) following centrifugation at 2000 rpm for 25 min. The intra-cellular staining has been described elsewhere [15 (link)]. Briefly, mononuclear cells were harvested and incubated with cell stimulation cocktail (eBioscience) in the presence of 2 μM of monensin (eBioscience) at a density of 1 × 10⁶ cells/ml overnight at 37 °C with 5% CO₂. Cells were stained with antibody against CD3, CD4 and CD8 and then fixed and permeabilized using permeabilization buffer (Biolegend) before intra-cellularly stained with anti-mouse IFN-γ, anti-Mouse Granzyme B, anti-Mouse Perforin or isotype-matched control mAb for 20 min in the dark at room temperature. Samples were analysed by flow cytometry using a FACS Calibur analyser (Becton Dickinson).