Blood samples and preparation
Venous blood samples were drawn into ethylenediaminetetraacetic acid tubes within 24 hours after the patient is diagnosed with sepsis, isovolumetric venous blood samples were collected from healthy volunteers in the physical examination center of our hospital. Blood samples were refrigerated at 4°C after EDTA anticoagulation. Density gradient centrifugation was conducted at 2,000 rpm for 20 minutes to isolate Peripheral blood mononuclear cells(PBMCs), which were used to detect the expression of membrane markers, and plasma, which was stored at -80°C for subsequent cytokine detection. The concentration of PBMCs was adjusted to 1 × 106/mL in RPMI 1640 culture solution supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM/L-glutamine, and 10% fetal calf serum (Gibco). The cell suspension was seeded onto 12-well cell culture plates. Cells were treated with 1ul Leukocyte Activation Cocktail, with BD GolgiPlug(BD), and incubated in darkness at 37°C under 5%-CO2 atmosphere for 6 hours.