Chapter 18 In Vitro Assay for Sensitive Determination of Human Blood PMN Responses Noah Fine, William Khoury, and Michael Glogauer Abstract 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 degranu- lation, 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. Key words PMNs, Flow cytometry, CD marker 1 Introduction PMNs are the most abundant leukocyte subset in human blood and act as critical first responders during acute inflammation [1]. They express a wide range of surface receptors to various target ligands, including: pathogen associated molecular patterns (PAMPs), cyto- kines, chemokines, and adhesion receptors. As an important aspect of their ability to quickly and effectively respond to a wide range of insults, PMNs display exquisite sensitivity to extracellular stimuli including PAMPs, cytokines, chemokines, mechanosensory stimuli, and other components of the innate and adaptive immune system, such as target bound antibody, complement proteins, macro- phages, and platelets. During priming and activation PMNs undergo degranulation [2], one consequence of which is the rapid surface upregulation of a number of Cluster of Differentiation (CD) markers, which in turn alter downstream PMN functionality. Due to the sensitivity of PMNs to in vitro manipulation [3–5], accurate flow cytometric determination of CD marker expression Mark T. Quinn and Frank R. DeLeo (eds.), Neutrophil: Methods and Protocols, Methods in Molecular Biology, vol. 2087, https://doi.org/10.1007/978-1-0716-0154-9_18, © Springer Science+Business Media, LLC, part of Springer Nature 2020 235