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Collection Highlights

Patch Clamp Electrophysiology: Methods and Protocols Mark

Dallas

Programmed Necrosis Methods and Protocols Adrian T. Ting

T Cell Development Methods and Protocols 1st Edition Rémy

Bosselut

Electrophoretic Separation of Proteins: Methods and

Protocols Biji T. Kurien
Redox-Mediated Signal Transduction: Methods and Protocols
John T. Hancock

Mitochondrial DNA Methods and Protocols 3rd Edition

Matthew Mckenzie (Eds.)

Plant Proteomics Methods and Protocols Methods in

Molecular Biology 2139 3rd Edition Jesus V. Jorrin-Novo

Animal Influenza Virus Methods and Protocols Methods in

Molecular Biology 2123 Band 2123 3rd Edition Erica
Spackman (Editor)

Zebrafish Methods and Protocols Koichi Kawakami

Volume 2087

Methods in Molecular Biology

Series Editor
John M. Walker
School of Life and Medical Sciences, University of Hertfordshire, Hatfield,
Hertfordshire, UK

For further volumes: http://​www.​springer.​com/​series/​7651

For over 35 years, biological scientists have come to rely on the
research protocols and methodologies in the critically
acclaimedMethods in Molecular Biology series. The series was the first
to introduce the step-by-step protocols approach that has become the
standard in all biomedical protocol publishing. Each protocol is
provided in readily-reproducible step-by-step fashion, opening with an
introductory overview, a list of the materials and reagents needed to
complete the experiment, and followed by a detailed procedure that is
supported with a helpful notes section offering tips and tricks of the
trade as well as troubleshooting advice. These hallmark features were
introduced by series editor Dr. John Walker and constitute the key
ingredient in each and every volume of theMethods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols
from the series are indexed in PubMed.
Editors
Mark T. Quinn and Frank R. DeLeo

Neutrophil
Methods and Protocols
3rd ed. 2020
Editors
Mark T. Quinn
Department of Microbiology and Immunology, Montana State
University, Bozeman, MT, USA

Frank R. DeLeo
Laboratory of Bacteriology, Rocky Mountain Laboratories Division of
Intramural Research, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Hamilton, MT, USA

ISSN 1064-3745 e-ISSN 1940-6029

Methods in Molecular Biology
ISBN 978-1-0716-0153-2 e-ISBN 978-1-0716-0154-9
https://doi.org/10.1007/978-1-0716-0154-9

© Springer Science+Business Media, LLC, part of Springer Nature 2020

This work is subject to copyright. All rights are reserved by the

Publisher, whether the whole or part of the material is concerned,
specifically the rights of translation, reprinting, reuse of illustrations,
recitation, broadcasting, reproduction on microfilms or in any other
physical way, and transmission or information storage and retrieval,
electronic adaptation, computer software, or by similar or dissimilar
methodology now known or hereafter developed.

The use of general descriptive names, registered names, trademarks,

service marks, etc. in this publication does not imply, even in the
absence of a specific statement, that such names are exempt from the
relevant protective laws and regulations and therefore free for general
use.

The publisher, the authors, and the editors are safe to assume that the
advice and information in this book are believed to be true and accurate
at the date of publication. Neither the publisher nor the authors or the
editors give a warranty, express or implied, with respect to the material
contained herein or for any errors or omissions that may have been
made. The publisher remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.

Cover Caption: Top row, from left to right: 1. Human neutrophil

swarming against a cluster of Candida albicans. 2. Aspergillus
fumigatus spores were patterned in a cluster and allowed to grow into
hyphae. 3. Sytox green staining showing neutrophil extracellular trap
release inside a human neutrophil swarm against C. albicans. 4. A
patterned cluster of C. albicans yeast. Images taken by Alex Hopke and
prepared for cover by Xiao Wang (Center for Engineering in Medicine,
Massachusetts General Hospital, Harvard Medical School, Charlestown,
MA).

This Humana imprint is published by the registered company Springer

Science+Business Media, LLC, part of Springer Nature.
The registered company address is: 233 Spring Street, New York, NY
10013, U.S.A.
Dedication
This volume is dedicated to Dr. Niels Borregaard (1954–2017) in
recognition of his extensive contributions to neutrophil cell biology,
especially in the understanding of neutrophil granule formation,
subcellular distribution, and function. Niels was a friend and
contributor to the first two editions of this book. This volume is also
dedicated to our families, who are tolerant of the time we spend
researching neutrophils.
Preface
Neutrophils (also known as polymorphonuclear neutrophils (PMNs) or
granulocytes) are the most abundant white cell in humans.
Granulocytes and/or granulocyte precursors normally comprise ~60%
of the nucleated cells in bone marrow and the bloodstream. Mature
neutrophils have a typical circulating half-life of 6–8 h in the blood and
then migrate through tissues for ~2–3 days. Their relatively short
lifespan is devoted largely to surveillance for invading microorganisms.
During infection, the neutrophil lifespan is extended, granulopoiesis
increases, and large numbers of neutrophils are rapidly recruited to the
site(s) of infection. Following recognition (binding) and phagocytosis of
microorganisms, neutrophils utilize an extraordinary array of oxygen-
dependent and oxygen-independent microbicidal weapons to destroy
infectious agents. Oxygen-dependent mechanisms involve the
production of reactive oxygen species (ROS), while oxygen-independent
mechanisms include degranulation and release of lytic enzymes and
bactericidal peptides. Inasmuch as these processes are highly effective
at killing most ingested microbes, neutrophils serve as the primary
cellular defense against infection.
The aim ofNeutrophils: Methods and Protocols, Third Edition, is to
provide (1) a set of protocols to assess basic neutrophil functions and
(2) protocols for investigating specialized areas in neutrophil research.
This volume is designed for the basic researcher involved in the study
of neutrophil function. A wide variety of methods have been developed
to assess neutrophil function, and these methods have been
instrumental in advancing our understanding of the role of neutrophils
in host defense and inflammatory disease. For those researchers and
clinicians interested in the study of neutrophils, the availability of a
comprehensive source of protocols describing the most modern
methodological advances in neutrophil biology is invaluable, as many
publications do not provide information on the finer details critical to
success of a given method. As such, we have compiled a series of
protocols written by leading researchers in the field that provide
detailed guidelines for establishing and performing the most common
neutrophil function assays. Hints of the best way to perform these
methods as well as guidance in detecting associated problems are
included, so novice investigators will also be able to effectively utilize
these assays.
In the third edition ofNeutrophils: Methods and Protocols , chapters
retained from previous editions have been updated and include many
new approaches. In addition, theThird Edition contains a number of
new chapters that were not included in the first two editions. Part I
contains overviews of neutrophil biology and function, and disorders of
neutrophils. Part II describes commonly used methods to isolate
neutrophils from humans and other animal species. This section also
contains a chapter that describes use of a neutrophil transplant model
with zebrafish larva. Part III details methods for investigating
chemotaxis, transmigration, phagocytosis, and bactericidal activity.
Three chapters provide methods used to assess neutrophil
transmigration, chemotaxis, or swarming against live microbes. One of
the chapters updated from theSecond Edition covers a neutrophil
microinjection approach for studying phagocytosis, and a new chapter
details use of imaging flow cytometry to evaluate phagocytosis. Several
of these chapters are new and contain innovative methods and
approaches for studying neutrophils. Part IV includes protocols that
measure neutrophil apoptosis, calcium signal transduction,
degranulation and detection of cytoplasmic granules, gene expression,
transcription factors, and apoptosis. Part V provides multiple assays for
measuring production of intracellular and/or extracellular reactive
oxygen species. In addition, there is a chapter that details the history
and use of the cell-free NADPH oxidase assay, an iconic assay for studies
of the neutrophil NADPH oxidase. Part VI provides chapters that
describe how to analyze formation and function of neutrophil
extracellular traps, including new chapters on visualization of NETs by
intravital microscopy and detection of NETs in tissues. In addition to
the step-by-step protocols, theNotes section of each chapter provides
an outstanding depot of useful and interesting information not typically
published in the Methods sections of standard journal articles.
We thank John M. Walker, Series Editor, and Springer Nature for the
opportunity to assemble an outstanding collection of chapters and for
help with the publication of the volume. We also thank the NIH IDeA
Program (COBRE Grant GM110732) and the NIH Intramural Research
Program, National Institutes of Allergy and Infectious Diseases, for
support. Finally, we thank the authors for taking time to write
outstanding chapters.
Mark T. Quinn
Frank R. DeLeo
Bozeman, MT, USA, Hamilton, MT, USA
Contents
Part I Neutrophils and Neutrophil Disorders: Overviews
1 The Role of Neutrophils in the Immune System:​An Overview
Harry L. Malech, Frank R. DeLeo and Mark T. Quinn
2 Neutrophil Defects and Diagnosis Disorders of Neutrophil
Function:​An Overview
Mary C. Dinauer
Part II Neutrophil Isolation
3 Isolation of Human Neutrophils from Venous Blood
Silvie Kremserova and William M. Nauseef
4 Isolation of Neutrophils from Nonhuman Species
Daniel W. Siemsen, Liliya N. Kirpotina, Natalia Malachowa,
Igor A. Schepetkin, Adeline R. Porter, Benfang Lei, Frank R. DeLeo
and Mark T. Quinn
5 Isolation of Neutrophils from Larval Zebrafish and Their
Transplantation into Recipient Larvae for Functional Studies
Hannah Darroch, Jonathan W. Astin and Christopher J. Hall
Part III Neutrophil Chemotaxis, Phagocytosis, and Bactericidal
Activity
6 Analysis of Neutrophil Transmigration Through Epithelial Cell
Monolayers
Liliya N. Kirpotina, Douglas J. Kominsky, Mark T. Quinn and
Steve D. Swain
7 Quantification of Chemotaxis or Respiratory Burst Using Ex Vivo
Culture-Derived Murine Neutrophils
Klaudia Szymczak, Margery G. H. Pelletier and Peter C. W. Gaines
8 Ex Vivo Human Neutrophil Swarming Against Live Microbial
Targets
Alex Hopke and Daniel Irimia
9 Microinjection and Micropipette-Controlled Phagocytosis
Methods for Neutrophils
 Maurice B. Hallett, Jennie S. Campbell, Iraj Laffafian and
Sharon Dewitt
10 Using Imaging Flow Cytometry to Quantify Neutrophil
Phagocytosis
Asya Smirnov, Michael D. Solga, Joanne Lannigan and Alison K. Criss
11 Visualization and Quantification of Phagocytosis by
Neutrophils
Gaelen Guzman and Fikadu G. Tafesse
12 Analysis of Neutrophil Bactericidal Activity
Nicholas J. Magon, Heather A. Parker, Louisa V. Ashby,
Reuben J. Springer and Mark B. Hampton
Part IV Biochemistry, Biology, and Signal Transduction of
Neutrophils
13 Assessment of Neutrophil Apoptosis
Nicole D. Barth, Marc Vendrell, David A. Dorward, Adriano G. Rossi
and Ian Dransfield
14 Optical Methods for the Measurement and Manipulation of
Cytosolic Calcium Signals in Neutrophils
Maurice B. Hallett, Rhiannon E. Roberts and Sharon Dewitt
15 Labeling Acidic Compartments of Neutrophils with Cresyl Violet
Philip P. Ostrowski, Ziv Roth and Sergio Grinstein
16 Neutrophil Degranulation of Azurophil and Specific Granules
Samia Bedouhène, Pham My-Chan Dang, Margarita Hurtado-
Nedelec and Jamel El-Benna
17 Influence of Oxygen on Function and Cholesterol Composition
of Murine Bone Marrow-Derived Neutrophils
Katja Branitzki-Heinemann, Graham Brogden and
Maren von Kö ckritz-Blickwede
18 In Vitro Assay for Sensitive Determination of Human Blood
PMN Responses
Noah Fine, William Khoury and Michael Glogauer
19 Fast and Accurate Quantitative Analysis of Cytokine Gene
Expression in Human Neutrophils by Reverse Transcription Real-
Time PCR
Nicola Tamassia, Marco A. Cassatella and Flavia Bazzoni
20 Detection of Intact Transcription Factors in Human Neutrophils
Patrick P. McDonald and Richard D. Ye
21 Genome-Scale Transcript Analyses of Human Neutrophils
Scott D. Kobayashi, Adeline R. Porter, Sarah L. Anzick,
Dan E. Sturdevant and Frank R. DeLeo
Part V NADPH Oxidase and Production of Reactive Oxygen Species
22 Measurement of Respiratory Burst Products, Released or
Retained, During Activation of Professional Phagocytes
Claes Dahlgren, Halla Bjö rnsdottir, Martina Sundqvist,
Karin Christenson and Johan Bylund
23 Cell-Free NADPH Oxidase Activation Assays:​A Triumph of
Reductionism
Edgar Pick
Part VI Analysis of Neutrophil Extracellular Traps
24 Immunofluorescen​t Detection of NET Components in Paraffin-
Embedded Tissue
Ulrike Abu-Abed and Volker Brinkmann
25 Detection, Visualization, and Quantification of Neutrophil
Extracellular Traps (NETs) and NET Markers
Nicole de Buhr and Maren von Kö ckritz-Blickwede
26 Imaging of Neutrophils and Neutrophil Extracellular Traps
(NETs) with Intravital (In Vivo) Microscopy
Iwona Cichon, Michal Santocki, Weronika Ortmann and
Elzbieta Kolaczkowska
Index
Contributors
Ulrike Abu-Abed
Microscopy Core Facility, Max Planck Institute for Infection Biology,
Berlin, Germany
Cellular Microbiology, Max Planck Institute for Infection Biology, Berlin,
Germany

Sarah L. Anzick
Genomics Unit, Rocky Mountain Laboratories, National Institute of
Allergy and Infectious Diseases, National Institutes of Health, Hamilton,
MT, USA

Louisa V. Ashby
Department of Pathology and Biomedical Science, Centre for Free
Radical Research, University of Otago Christchurch, Christchurch, New
Zealand

Jonathan W. Astin
Department of Molecular Medicine and Pathology, Faculty of Medical
and Health Sciences, University of Auckland, Auckland, New Zealand

Nicole D. Barth
Centre for Inflammation Research, Queen’s Medical Research Institute,
University of Edinburgh, Edinburgh, UK

Flavia Bazzoni
Department of Medicine, Section of General Pathology, University of
Verona, Verona, Italy

Samia Bedouhène
Centre de Recherche sur l’Inflammation (CRI), INSERM-U1149, CNRS-
ERL8252, Laboratoire d’Excellence Inflamex, Université Paris Diderot-
Sorbonne Paris Cité, Faculté de Médecine, Site Xavier Bichat, Paris,
France
Laboratoire de Biochimie Analytique et de Biotechnologie, Faculté des
Sciences Biologiques et des Sciences Agronomiques, Université
Mouloud Mammeri de Tizi-Ouzou, Tizi Ouzou, Algeria
Halla Björnsdottir
Department of Oral Microbiology and Immunology, Institute of
Odontology, Sahlgrenska Academy at University of Gothenburg,
Gothenburg, Sweden

Katja Branitzki-Heinemann
Department of Physiological Chemistry, University of Veterinary
Medicine Hannover, Hannover, Germany
Research Center for Emerging Infections and Zoonoses (RIZ),
University of Veterinary Medicine Hannover, Hannover, Germany

Volker Brinkmann
Microscopy Core Facility, Max Planck Institute for Infection Biology,
Berlin, Germany

Graham Brogden
Department of Physiological Chemistry, University of Veterinary
Medicine Hannover, Hannover, Germany

Johan Bylund
Department of Oral Microbiology and Immunology, Institute of
Odontology, Sahlgrenska Academy at University of Gothenburg,
Gothenburg, Sweden

Jennie S. Campbell
Neutrophil Signalling Group, School of Dentistry, Cardiff University,
Cardiff, UK

Marco A. Cassatella
Department of Medicine, Section of General Pathology, University of
Verona, Verona, Italy

Karin Christenson
Department of Oral Microbiology and Immunology, Institute of
Odontology, Sahlgrenska Academy at University of Gothenburg,
Gothenburg, Sweden

Iwona Cichon
Department of Experimental Hematology, Institute of Zoology and
Biomedical Research, Jagiellonian University, Krakó w, Poland

Alison K. Criss
Department of Microbiology, Immunology, and Cancer Biology,
University of Virginia, Charlottesville, VA, USA

Claes Dahlgren
Department of Rheumatology and Inflammation Research, Institute of
Medicine, Sahlgrenska Academy at University of Gothenburg,
Gothenburg, Sweden

Pham My-Chan Dang

Centre de Recherche sur l’Inflammation (CRI), INSERM-U1149, CNRS-
ERL8252, Laboratoire d’Excellence Inflamex, Université Paris Diderot-
Sorbonne Paris Cité, Faculté de Médecine, Site Xavier Bichat, Paris,
France

Hannah Darroch
Department of Molecular Medicine and Pathology, Faculty of Medical
and Health Sciences, University of Auckland, Auckland, New Zealand

Nicole de Buhr
Department of Physiological Chemistry, University of Veterinary
Medicine Hannover, Hannover, Germany
Research Center for Emerging Infections and Zoonoses (RIZ),
University of Veterinary Medicine Hannover, Hannover, Germany

Frank R. DeLeo
Laboratory of Bacteriology, Rocky Mountain Laboratories, Division of
Intramural Research, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Hamilton, MT, USA

Sharon Dewitt
School of Biochemistry, University of Bristol, Bristol, UK
School of Medicine and School of Dentistry, Cardiff University, Cardiff,
UK
Mary C. Dinauer
Department of Pediatrics (Hematology/Oncology), St. Louis Children’s
Hospital, Washington University School of Medicine in St. Louis, St.
Louis, MO, USA
Department of Pathology and Immunology, St. Louis Children’s
Hospital, Washington University School of Medicine in St. Louis, St.
Louis, MO, USA

David A. Dorward
Centre for Inflammation Research, Queen’s Medical Research Institute,
University of Edinburgh, Edinburgh, UK

Ian Dransfield
Centre for Inflammation Research, Queen’s Medical Research Institute,
University of Edinburgh, Edinburgh, UK

Jamel El-Benna
Centre de Recherche sur l’Inflammation (CRI), INSERM-U1149, CNRS-
ERL8252, Laboratoire d’Excellence Inflamex, Université Paris Diderot-
Sorbonne Paris Cité, Faculté de Médecine, Site Xavier Bichat, Paris,
France

Noah Fine
Faculty of Dentistry, University of Toronto, Toronto, ON, Canada

Peter C. W. Gaines
Department of Biological Sciences, University of Massachusetts Lowell,
Lowell, MA, USA

Michael Glogauer
Faculty of Dentistry, University of Toronto, Toronto, ON, Canada
Department of Dental Oncology, Maxillofacial and Ocular Prosthetics,
Princess Margaret Cancer Centre, Toronto, ON, Canada
Centre for Advanced Dental Research and Care, Mount Sinai Hospital,
Toronto, ON, Canada

Sergio Grinstein
Program in Cell Biology, Hospital for Sick Children, Toronto, ON, Canada
Department of Biochemistry, University of Toronto, Toronto, ON,
Canada

Gaelen Guzman
Department of Molecular Microbiology and Immunology, Oregon Health
and Science University, Portland, OR, USA

Christopher J. Hall
Department of Molecular Medicine and Pathology, Faculty of Medical
and Health Sciences, University of Auckland, Auckland, New Zealand

Maurice B. Hallett
Neutrophil Signalling Group, School of Medicine, Cardiff University,
Cardiff, UK

Mark B. Hampton
Department of Pathology and Biomedical Science, Centre for Free
Radical Research, University of Otago Christchurch, Christchurch, New
Zealand

Alex Hopke
Center for Engineering in Medicine, Massachusetts General Hospital,
Harvard Medical School, Shriners Hospital for Children, Boston, MA,
USA

Margarita Hurtado-Nedelec
Centre de Recherche sur l’Inflammation (CRI), INSERM-U1149, CNRS-
ERL8252, Laboratoire d’Excellence Inflamex, Université Paris Diderot-
Sorbonne Paris Cité, Faculté de Médecine, Site Xavier Bichat, Paris,
France
AP-HP, Centre Hospitalier Universitaire Xavier Bichat, UF
Dysfonctionnements Immunitaires, Paris, France

Daniel Irimia
Center for Engineering in Medicine, Massachusetts General Hospital,
Harvard Medical School, Shriners Hospital for Children, Boston, MA,
USA
William Khoury
Faculty of Dentistry, University of Toronto, Toronto, ON, Canada

Liliya N. Kirpotina
Department of Microbiology and Immunology, Montana State
University, Bozeman, MT, USA

Scott D. Kobayashi
Laboratory of Bacteriology, Rocky Mountain Laboratories, National
Institute of Allergy and Infectious Diseases, National Institutes of
Health, Hamilton, MT, USA

Elzbieta Kolaczkowska
Department of Experimental Hematology, Institute of Zoology and
Biomedical Research, Jagiellonian University, Krakó w, Poland

Douglas J. Kominsky
Department of Microbiology and Immunology, Montana State
University, Bozeman, MT, USA

Silvie Kremserova
Inflammation Program and Department of Medicine, Roy J. and Lucille
A. Carver College of Medicine, University of Iowa, Iowa City, IA, USA

Iraj Laffafian
Neutrophil Signalling Group, School of Medicine, Cardiff University,
Cardiff, UK

Joanne Lannigan
Department of Microbiology, Immunology, and Cancer Biology,
University of Virginia, Charlottesville, VA, USA

Benfang Lei
Department of Microbiology and Immunology, Montana State
University, Bozeman, MT, USA

Nicholas J. Magon
Department of Pathology and Biomedical Science, Centre for Free
Radical Research, University of Otago Christchurch, Christchurch, New
Zealand

Natalia Malachowa
Laboratory of Bacteriology, Rocky Mountain Laboratories, Division of
Intramural Research, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Hamilton, MT, USA

Harry L. Malech
Laboratory of Clinical Immunology and Microbiology, Division of
Intramural Research, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD, USA

Patrick P. McDonald
Pulmonary Division, Medicine Faculty, Université de Sherbrooke,
Sherbrooke, QC, Canada

William M. Nauseef
Inflammation Program and Department of Medicine, Roy J. and Lucille
A. Carver College of Medicine, University of Iowa, Iowa City, IA, USA

Weronika Ortmann
Department of Experimental Hematology, Institute of Zoology and
Biomedical Research, Jagiellonian University, Krakó w, Poland

Philip P. Ostrowski
Program in Cell Biology, Hospital for Sick Children, Toronto, ON, Canada
Department of Biochemistry, University of Toronto, Toronto, ON,
Canada

Heather A. Parker
Department of Pathology and Biomedical Science, Centre for Free
Radical Research, University of Otago Christchurch, Christchurch, New
Zealand

Margery G. H. Pelletier
Department of Biological Sciences, University of Massachusetts Lowell,
Lowell, MA, USA

Edgar Pick
Department of Clinical Microbiology and Immunology, Sackler School of
Medicine, Tel Aviv University, Tel Aviv, Israel

Adeline R. Porter
Laboratory of Bacteriology, Rocky Mountain Laboratories, Division of
Intramural Research, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Hamilton, MT, USA

Mark T. Quinn
Department of Microbiology and Immunology, Montana State
University, Bozeman, MT, USA

Rhiannon E. Roberts
Neutrophil Signalling Group, Cardiff, UK

Adriano G. Rossi
Centre for Inflammation Research, Queen’s Medical Research Institute,
University of Edinburgh, Edinburgh, UK

Ziv Roth
Program in Cell Biology, Hospital for Sick Children, Toronto, ON, Canada

Michal Santocki
Department of Experimental Hematology, Institute of Zoology and
Biomedical Research, Jagiellonian University, Krakó w, Poland

Igor A. Schepetkin
Department of Microbiology and Immunology, Montana State
University, Bozeman, MT, USA

Daniel W. Siemsen
Department of Microbiology and Immunology, Montana State
University, Bozeman, MT, USA
Asya Smirnov
Department of Microbiology, Immunology, and Cancer Biology,
University of Virginia, Charlottesville, VA, USA

Michael D. Solga
UVA Flow Cytometry Core, University of Virginia, Charlottesville, VA,
USA

Reuben J. Springer
Department of Pathology and Biomedical Science, Centre for Free
Radical Research, University of Otago Christchurch, Christchurch, New
Zealand

Dan E. Sturdevant
Genomics Unit, Rocky Mountain Laboratories, National Institute of
Allergy and Infectious Diseases, National Institutes of Health, Hamilton,
MT, USA

Martina Sundqvist
Department of Rheumatology and Inflammation Research, Institute of
Medicine, Sahlgrenska Academy at University of Gothenburg,
Gothenburg, Sweden

Steve D. Swain
Department of Microbiology and Immunology, Montana State
University, Bozeman, MT, USA

Klaudia Szymczak
Department of Biological Sciences, University of Massachusetts Lowell,
Lowell, MA, USA

Fikadu G. Tafesse
Department of Molecular Microbiology and Immunology, Oregon Health
and Science University, Portland, OR, USA

Nicola Tamassia
Department of Medicine, Section of General Pathology, University of
Verona, Verona, Italy
Marc Vendrell
Centre for Inflammation Research, Queen’s Medical Research Institute,
University of Edinburgh, Edinburgh, UK

Maren von Köckritz-Blickwede

Department of Physiological Chemistry, University of Veterinary
Medicine Hannover, Hannover, Germany
Research Center for Emerging Infections and Zoonoses (RIZ),
University of Veterinary Medicine Hannover, Hannover, Germany

Richard D. Ye
Kobilka Institute of Innovative Drug Discovery, School of Life and
Health Sciences, The Chinese University of Hong Kong, Shenzhen, China
Part I
Neutrophils and Neutrophil Disorders:
Overviews
© Springer Science+Business Media, LLC, part of Springer Nature 2020
M. T. Quinn, F. R. DeLeo (eds.), Neutrophil, Methods in Molecular Biology 2087
https://doi.org/10.1007/978-1-0716-0154-9_1

1. The Role of Neutrophils in the

Immune System: An Overview
Harry L. Malech1, Frank R. DeLeo2 and Mark T. Quinn3

(1) Laboratory of Clinical Immunology and Microbiology, Division of

Intramural Research, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD, USA
(2) Laboratory of Bacteriology, Rocky Mountain Laboratories, Division
of Intramural Research, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Hamilton, MT, USA
(3) Department of Microbiology and Immunology, Montana State
University, Bozeman, MT, USA

Mark T. Quinn
Email: mquinn@montana.edu

Abstract
Neutrophils, also known as polymorphonuclear neutrophils (PMNs),
have long been considered as the short-lived, nonspecific white cells
that form pus—and also happen to kill invading microbes. Indeed,
neutrophils were often neglected (and largely not considered) as
immune cells. This historic view of neutrophils has changed
considerably over the past several decades, and we now know that in
addition to playing the predominant role in the clearance of bacteria
and fungi, they have a major role in shaping the host response to
infection and immune system homeostasis. The change in our view of
the role of neutrophils in the immune system has been due in large part
to the study of these cells in vitro. Such work has been made possible
by new and/or improved methods and approaches used to investigate
neutrophils. These methods are the focus of this volume.

Key words Polymorphonuclear leukocyte – Granulocyte – Neutrophil

methods

1 Introduction
This valuable and unique book contains a compendium of methods and
reviews that does much more than allow one to study the biology of
neutrophils . What makes this collection of contributions so special is
that it highlights and facilitates using the neutrophil as a simple, pure,
single primary cell suspension model to study a remarkable array of
generalized cellular functions (e.g., adhesion, chemotaxis and
transmigration , phagocytosis , degranulation , oxygen radical
production, apoptosis , and gene expression ), as well as specialized
functions (e.g., formation of extracellular traps) and molecules
important to host defense against infection and the mediation and
resolution of inflammation (see Fig. 1). Consideration of the array of
chapter topics evokes some of the past history of inquiry into how
neutrophils function and how we evolved into the current widespread
use of the neutrophil as a convenient model system for studying so
many types of cellular processes and biochemical pathways.
Fig. 1 Illustration of key neutrophil functions. Note that for production reactive
oxygen species, secretion of granule components, and production of cytokines and
chemokines, only a few representative molecules are shown. HNE human neutrophil
elastase, IL-8 interleukin-8, IL-4 interleukin-4, LF lactoferrin, MPO myeloperoxidase ,
TNF-α tumor necrosis factor-α
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