Ficoll hypaque 10 771

First, the peripheral blood was diluted and mixed with RPIM1640 (Gibco, Germany) at a ratio of 1:1, and then gently layered on top of 4 mL of Ficoll-Hypaque 10,771 (Sigma, USA). The tubes were centrifugated at 1600 rpm for 30 min at 25 °C. Then, the PBMC layer was aspirated and transferred into a new fresh 15 mL conical centrifuge tube. The PBMCs were mixed with RPIM1640 to wash twice at 1300 rpm for 10 min. After cell counting, fresh PBMCs were stained with different FCM antibodies and tested using a BD Canto II Flow Cytometry System. The remaining PBMCs were resuspended and cryopreserved with freezing media, including 10% dimethysulfoxide (DMSO) and 90% fetal bovine serum. The cell number was generally at least 5 × 10⁶ per tube. Lastly, the frozen PBMC samples were transported into a liquid nitrogen container for long-time preservation after being stored in the freezing container at − 80 °C overnight. 5–10 samples were processed at a time and the subsequent freez thaw process followed every step of the protocol strictly.