Isolation of PBMCs from Venous Blood

Venous blood samples were collected in heparin tubes, and PBMCs were isolated using Ficoll gradient centrifugation, as previously described in^{64 (link)}. Whole blood was transferred to conical tubes and centrifuged at 2000 × g for 5 min. Plasma samples were then collected, and the remaining blood was diluted with RPMI (Corning, Manassas, VA, USA). Diluted blood was then layered over an appropriate volume of Histopaque (Histopaque-1077 Cell Separation Medium, Sigma Aldrich, St. Louis, MO, USA) and centrifuged for 20 min at 500 × g at room temperature without brake. The PBMC layer was carefully removed, transferred to a fresh tube, and washed with RPMI. Cells were stained and counted using Automated Cell Counter (BioRad, Hercules, California, USA). Isolated PBMCs were then cryopreserved in a freezing medium containing 10% dimethyl sulfoxide (DMSO) (MP Biomedicals, LLC, Irvine, CA, USA) and 90% heat-inactivated fetal bovine serum (FBS) and stored in liquid nitrogen for further use.