Isolation and Culture of PBMCs

PBMCs were isolated from fresh EDTA blood. Venous blood was collected in EDTA tubes (S-Monovette, Sarstedt, Sarstedt, Germany) and directly used for density gradient centrifugation. A total of 6 mL of blood was carefully layered on 6 mL of Lympholyte-poly Cell Separation Medium (Cedarlane, Burlington, ON, Canada). Samples were centrifuged for 35 min at 500 g without a break at room temperature. The PBMC (upper) layer was transferred to a new tube. After washing twice with PBS, the cells were counted and seeded at a concentration of 5 × 10⁵ cells/mL in an RPMI 1640 medium with 2% autologous plasma [28 (link)]. Experiments were performed at 37 °C (5% CO₂, humidified atmosphere).