PBMCs and splenocytes were isolated at each time point after sepsis induction. PBMCs were isolated using the Ficoll gradient method [27 (link)]. Isolated PBMCs were stimulated with LPS to observe and compare the levels of immune paralysis. Tumor necrosis factor (TNF)-α levels were measured 5 h after LPS stimulation. 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 levels were analyzed using a TNF-α enzyme-linked immunosorbent assay (ELISA) kit (ab236712, Abcam, Cambridge, MA, USA).
Splenocytes were isolated and stimulated with LPS to compare immune paralysis [28 (link)]. TNF-α levels were measured 5 h after LPS stimulation. Isolated splenocytes were seeded at a density of 5 × 105 cells/mL in 6-well plates, and 1 μg/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 was analyzed using a TNF-α ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA).
Splenocytes were isolated and stimulated with LPS to compare immune paralysis [28 (link)]. TNF-α levels were measured 5 h after LPS stimulation. Isolated splenocytes were seeded at a density of 5 × 105 cells/mL in 6-well plates, and 1 μg/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 was analyzed using a TNF-α ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA).
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