Isolation and Cryopreservation of Peripheral Blood Mononuclear Cells (PBMCs)

Whole blood was diluted with phosphate-buffered saline (PBS), and PBMC isolation was performed by density gradient centrifugation using Ficoll-Paque (GE Healthcare, IL, USA). The PBMC fraction was collected and washed three times with PBS. The cells were resuspended in RPMI 1640 Medium (Dutch modification) (Thermo Fisher Scientific, MA, USA) supplemented with 1 mM sodium pyruvate (Thermo Fisher Scientific), 2 mM GlutaMAX supplement (Thermo Fisher Scientific), and 5 µg/mL gentamicin (Centraform, Netherlands). For DNA methylation, single cell RNA sequencing (scRNA-seq), and single cell ATAC sequencing (scATAC-seq) analysis, and future stimulation assays, cells were cryopreserved in RPMI containing 40% bovine calf serum (Cytiva, MA, USA) and 15% dimethyl sulfoxide (VWR, PA, USA).