Ficoll hypaque density

Peripheral blood mononuclear cells (PBMCs) were isolated from model rats by Ficoll-Hypaque density gradient centrifugation (TBD Science, Tianjin, China). The cells were washed three times with sterile PBS, suspended in a concentration of 2 × 10⁶ cells/mL in RPMI 1640 (HyClone, Logan, UT, United States) supplemented with 10% heat-inactivated fetal calf serum and 1% penicillin-streptomycin (HyClone), and cultured at 37 °C in a CO₂ incubator. These cells were treated with 0.25 μmol/L DAPT (HY-13027; MedChem Express, Monmouth Junction, NJ, United States) and 0.25 mmol/L TGF-β inhibitor (LY-364947; MedChem Express), with 0.1% dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO, United States) as a control. After treatment for 24 h, all cells were collected for total RNA and protein extraction.

A total of 45–60 mL of peripheral venous blood was extracted from each PV patient and healthy control. PBMCs were isolated using the Ficoll–Hypaque density centrifugation (TBD Science, Tianjin, China). Positive selection was utilised to isolate CD4+ T cells with the EasySep Human CD4+ T Cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada) following the manufacturer’s protocol.