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 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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Borregaard N (2010) Neutrophils, from marrow to microbes. Immunity 33:657–670
Fine N, Barzilay O, Sun C et al (2019) Primed PMNs in healthy mouse and human circulation are first responders during acute inflammation. Blood Adv 3:1622–1637
Fearon DT, Collins LA (1983) Increased expression of C3b receptors on polymorphonuclear leukocytes induced by chemotactic factors and by purification procedures. J Immunol 130:370–375
Macey MG, McCarthy DA, Vordermeier S et al (1995) Effects of cell purification methods on CD11b and L-selectin expression as well as the adherence and activation of leucocytes. J Immunol Methods 181:211–219
Hamblin A, Taylor M, Bernhagen J et al (1992) A method of preparing blood leucocytes for flow cytometry which prevents upregulation of leucocyte integrins. J Immunol Methods 146:219–228
Oveisi M, Shifman H, Fine N et al (2019) Novel assay to characterize neutrophil responses to oral biofilms. Infect Immun 87(2).
Fine N, Hassanpour S, Borenstein A et al (2016) Distinct oral neutrophil subsets define health and periodontal disease states. J Dent Res 95:931–938
Lakschevitz FS, Hassanpour S, Rubin A et al (2016) Identification of neutrophil surface marker changes in health and inflammation using high-throughput screening flow cytometry. Exp Cell Res 342:200–209
Fine N, Sheikh Z, Al-Jaf F et al (2019) Differential response of human blood leukocytes to brushite, monetite, and calcium polyphosphate biomaterials. J Biomed Mater Res B Appl Biomater.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Fine, N., Khoury, W., Glogauer, M. (2020). In Vitro Assay for Sensitive Determination of Human Blood PMN Responses. In: Quinn, M., DeLeo, F. (eds) Neutrophil. Methods in Molecular Biology, vol 2087. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0154-9_18
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0154-9_18
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0153-2
Online ISBN: 978-1-0716-0154-9
eBook Packages: Springer Protocols