Ficoll paque separation medium

The peripheral blood of healthy persons (volunteers in our laboratory) was put into anticoagulant tubes. After 1:1 dilution with normal saline, the blood was slowly spread on the surface of the appropriate Ficoll-Paque separation medium (GE). Centrifugation was performed at 800 × g for 20 min. The white film was carefully absorbed after stratification. Centrifugation at 800 g for 5 min. Precipitation of human peripheral blood mononuclear cells. CD8⁺ T lymphocytes were isolated from human peripheral blood monocytes with a magnetic bead sorting kit (Miltenyi). In vitro expansion was carried out in TexMACS medium (Miltenyi) containing hIL2 (50 U/mL, Miltenyi) and hIL-7-FC (70 ng/mL, Miltenyi) in a 5% CO₂ incubator at 37 °C. For the specific steps, please refer to the previous publication of our research group [12 (link)]. KYSE150 cells were conventionally cultured in RPMI 1640 medium (containing 10% FBS, Gibco). When the confluence was 60–70%, CD8⁺ T lymphocytes were added to the cell culture medium at a ratio of 10:1 CD8⁺ T lymphocytes: KYSE150 cells and cocultured at 37 °C in a 5% CO₂ incubator.

Blood samples were collected in lithium heparin tubes and PBMCs were freshly isolated by density centrifugation using Ficoll-Paque separation medium (GE Healthcare, Sweden). 2 × 10⁶ PBMCs per ml of RPMI, supplemented with 10% FCS and 250 U/ml penicillin, 250 μg/ml streptomycin, were cultured at 37°C, and stimulated fresh with 100 IU or 1000 IU pegylated IFN α-2a (Roche, Switzerland) for 2 and 4 hours. PBMCs were immediately lysed in Trizol reagent (Invitrogen, USA) following stimulation, thus preserving the RNA and stored at -80°C until analysis.