In Vitro Assay for Sensitive Determination of Human Blood PMN Responses.

Polymorphonuclear neutrophils (PMNs) are the most common leukocytes in the circulation and exhibit a wide range of distinct cellular phenomena as part of their microbicidal killing activities, including degranulation, phagocytosis, reactive oxygen species (ROS) production, adhesion, chemotaxis and production of PMN Extracellular Traps (NETs). As a simple in vitro test of PMN functional responses in human blood we have developed a multicolor flow cytometry-based assay of PMN cluster of differentiation (CD) surface marker expression. Short incubations of whole human blood can be performed in the presence of a wide range of agonists or inhibitors, followed by sensitive detection of changes in CD marker expression. This protocol has the advantage that small amounts of human blood are necessary and there are no PMN isolation steps, which can alter PMN activation status.