PREPARATION AND FUNCTIONAL ANALYSIS OF OTHER HUMAN LYMPHOID AND NONLYMPHOID CELLS-Isolation and Functional Analysis of Human Neutrophils

摘要

This unit describes the isolation of human polymorphonuclear neutrophils (PMN) from blood using dextran sedimentation and Percoll or Ficoll-Paque density gradients. Assays of neutrophil functions including respiratory burst activation, phagocytosis, and microbial killing are also described. Human polymorphonuclear neutrophils (PMN) are the most abundant nucleated cell in circulating blood (2 to 8 x 10~6 per ml). PMN possess characteristic cytoplasmic granules containing antimicrobial polypeptides and can produce antimicrobial reactive oxygen species (ROS, e.g., H_2O_2 and hypochlorous acid). The life cycle of the PMN includes maturation in and release from the bone marrow, circulation in blood, loose adherence to vascular endothelia, and clearance of senescent PMN by the reticuloen-dothelial system. If activated, peripheral PMN adhere to vascular endothelia and migrate out of the vasculature, moving in the direction of infectious or inflammatory stimuli, a process that significantly alters their behaviors (Zarember and Kuhns, 2011). Upon encountering microbes, PMN may form neutrophil extracellular traps (NETs), release the contents of their granules, produce inflammatory mediators, produce ROS, and engulf microbes through phagocytosis. Following activation, PMN can continue to contribute to the evolving inflammatory response or undergo apoptosis, usually culminating in the phagocytosis of the PMN themselves by tissue macrophages.