Simultaneous Measurement of Changes in Neutrophil Granulocyte Membrane Potential, Intracellular pH, and Cell Size by Multiparametric Flow Cytometry

Alexander Elias Paul Stratmann,Lisa Wohlgemuth,Maike Elisabeth Erber,Karl Josef Föhr,Stefan Hug,Markus Stefan Huber-Lang,Adam Omar Khalaf Mohamed,Stefan Bernhard,Laura Vidoni,David Alexander Christian Messerer,Michael Fauler,Frederik Münnich,Marco Mannes,Bertram Dietrich Thomaß,Laura Stukan

doi:10.3390/biomedicines9111504

Alexander Elias Paul Stratmann, Lisa Wohlgemuth + Show 13 more

Open Access

https://doi.org/10.3390/biomedicines9111504

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Journal: Biomedicines	Publication Date: Oct 20, 2021
Citations: 3	License type: CC BY 4.0

Affiliation: University Hospital Ulm, University of Ulm

Abstract

Neutrophils provide rapid and efficient defense mechanisms against invading pathogens. Upon stimulation with proinflammatory mediators, including complement factors and bacterial peptides, neutrophils respond with changes in their membrane potential, intracellular pH, and cellular size. This study provides an approach to quantify these important changes simultaneously using multiparametric flow cytometry, thereby revealing a typical sequence of neutrophil activation consisting of depolarization, alkalization, and increase in cellular size. Additionally, the time resolution of the flow cytometric measurement is improved in order to allow changes that occur within seconds to be monitored, and thus to enhance the kinetic analysis of the neutrophil response. The method is appropriate for the reliable semiquantitative detection of small variations with respect to an increase, no change, and decrease in those parameters as demonstrated by the screening of various proinflammatory mediators. As a translational outlook, the findings are put into context in inflammatory conditions in vitro as well as in a clinically relevant whole blood model of endotoxemia. Taken together, the multiparametric analysis of neutrophil responsiveness regarding depolarization, alkalization, and changes in cellular size may contribute to a better understanding of neutrophils in health and disease, thus potentially yielding innovative mechanistic insights and possible novel diagnostic and/or prognostic approaches.

Highlights

Licensee MDPI, Basel, Switzerland.Neutrophil granulocytes represent quantitatively the most relevant cell type of the innate immunity
When this decline is not taken into account, results may vary because control and stimulated cells cannot be measured in parallel by the same flow cytometer
The approach for compensation as described in the method section could address this issue as confirmed in Figure 1e, which shows that the measurement of three different tubes with control or stimulated neutrophils yield comparable results

Summary

Introduction

Licensee MDPI, Basel, Switzerland.Neutrophil granulocytes (neutrophils) represent quantitatively the most relevant cell type of the innate immunity. Neutrophils advance to the site of inflammation by chemotaxis, where they exert several typical physiological functions to combat invading pathogens, including the generation of reactive oxygen species (ROS), phagocytosis, and the formation of neutrophil extracellular traps [1,2,4,6]. In the context of systemic inflammation, such as after severe trauma or during sepsis, neutrophils are extensively stimulated [3,7,8,9]. In this regard, excessive neutrophil activity contributes to organ dysfunction and mortality [3,4,10]. Prolonged inflammation may result in paralysis of the neutrophils’

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