Isolation of Human venous blood leukocytes
There were 16 healthy volunteers, aged 26±5 years, whose blood collection was approved by the Medical Ethics Committee of Changchun Jiahe Surgical Hospital. We then used sucrose density-gradient centrifugation (endotoxin < 0.1EU, Tianjin Haoyang Huake Biotechnology Co., Ltd.) to separate white blood cells (WBCs) from venous blood. The samples were collected at room temperature and centrifuged at 1800 rpm for 25 minutes. The mononuclear cell layer, comprised of mostly lymphocytes with a few monocytes, and the multinuclear cell layer, comprised of mostly neutrophils with few contaminating mononuclear cells, were taken out to be mixed. Any contaminating erythrocytes were lysed and washed out. 1640 medium containing 10% FBS was used for resuspension. The morphology of the cells was observed with leukocyte staining solution, and the density was adjusted to 1×106 cells/ml. Blood samples from different volunteers were collected and used for each experiment in order to eliminate individual differences and ensure the study’s repeatability.