Ficoll lymphocyte separation medium

Blood samples were collected from healthy donors, an equal volume of PBS was added. Ficoll lymphocyte separation medium (TBD Science, LTS1077, Tianjin, China) was used to isolate the peripheral blood mononuclear cells (PBMCs). After PBMC separation, CD8 immunomagnetic beads (20 μl/10⁷ cells) purchased from Miltenyi (Germany) were added and incubated at 4 °C for 15 min. The MS column was prewashed and used to separate CD8⁺T cells. Before the activation of T cells, cytokines (Human Recombinant IL-2, Peprotech) 20 ng/ml were added to ImmunoCult™-XF T Cell Expansion Medium (Stemcell Technologies, Canada) and mix thoroughly. To activate T cells, add 25 µl/ml of ImmunoCult™ Human CD3/CD28 T Cell Activator(Stemcell Technologies, Canada) to the cell suspension. Incubate cells at 37 °C and 5% CO₂ for up to 3 days. After 3 days of activation, the cells were further cultured 7–10 days for further experiments.

Blood sample were collected from mice 12 hours after CLP operation as described previously. The peripheral blood mononuclear cells (PBMC) were collected using Ficoll lymphocyte separation medium (TBD Science, Tianjin, China). Total protein of PBMC was extracted using Radio Immunoprecipitation Assay kits (Beyotime Biotechnology Company, Jiangsu, China) according to the manufacturer's instruction. Cytoplasmic and nuclear protein were extracted with NE-PER Nuclearand Cytoplasmic Extraction Reagents (Pierce Biotechnology) according to the manufacturer's instruction. The protein concentrations were determined by BCA protein assay kit (Beyotime Biotechnology Company, Jiangsu, China). Then protein extracts were fractionated in 10% polyacrylamide sodium dodecyl sulfate gels and transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% fat-free milk in 0.1% Tween 20 tris-buffered saline (TBST) buffer for 2 hours and then was incubated with antibody against β-actin (1 : 1000), NF-κb (1 : 1000), and lamin B1 (1 : 1000) overnight at 4°C. Then, the membrane was incubated with horseradish peroxidase (HRP) conjugated secondary antibody for 1 hour. An ECL chemiluminescence system was used to visualize the binding and images were developed using UVP imaging system.