2.8. Ex vivo Peripheral Blood Mononuclear Cell (PBMC) Stimulation with LPS

PBMCs were isolated using the Ficoll gradient method, as previously described [7]. Isolated PBMCs were stimulated with LPS to observe and compare the level of immune paralysis. TNF-α levels were measured 5 h after LPS stimulation. In brief, isolated PBMCs were seeded at a density of 1 × 105 cells/mL in 96-well plates, and 100 ng/mL LPS (Escherichia coli O111: B4; Sigma-Aldrich, St. Louis, MO, USA) was added to each well. After 5 h, the culture medium was collected, and TNF-α level was analyzed using a TNF-α ELISA kit (ab236712, Abcam, MA, USA).