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.