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.