International Journal of

Molecular Sciences

Review
Methods for the Assessment of NET Formation: From
Neutrophil Biology to Translational Research
Marina Stoimenou † , Georgios Tzoros † , Panagiotis Skendros * and Akrivi Chrysanthopoulou *

Laboratory of Molecular Hematology, Department of Medicine, Democritus University of Thrace,

68100 Alexandroupolis, Greece
* Correspondence: pskendro@med.duth.gr (P.S.); akrivika@hotmail.com (A.C.)
† These authors contributed equally to this work.

Abstract: Several studies have indicated that a neutrophil extracellular trap (NET) formation, apart
from its role in host defense, can contribute to or drive pathogenesis in a wide range of inflammatory
and thrombotic disorders. Therefore, NETs may serve as a therapeutic target or/and a diagnostic
tool. Here, we compare the most commonly used techniques for the assessment of NET formation.
Furthermore, we review recent data from the literature on the application of basic laboratory tools
for detecting NET release and discuss the challenges and the advantages of these strategies in NET
evaluation. Taken together, we provide some important insights into the qualitative and quantitative
molecular analysis of NETs in translational medicine today.

Keywords: neutrophil; neutrophil extracellular traps; techniques; tools

1. Introduction
Citation: Stoimenou, M.; Tzoros, G.;
Neutrophils represent the most abundant innate immune cells and are the first to
Skendros, P.; Chrysanthopoulou, A.
migrate to sites of tissue inflammation. Their half-life in circulation varies from 6 h to a few
Methods for the Assessment of NET
days. Thus, at steady-state conditions, renewal and maintenance of the neutrophil pool
Formation: From Neutrophil Biology
to Translational Research. Int. J. Mol.
are ensured by constant bone marrow granulopoiesis. However, during systemic inflam-
Sci. 2022, 23, 15823. https://doi.org/
matory conditions, a reprogramming of the hematopoietic response, named emergency
10.3390/ijms232415823
granulopoiesis, leads to the commencement of de novo generation of high numbers of
neutrophils from myeloid progenitors and their mobilization to circulation [1].
Academic Editors: Kim M.
Neutrophils contain a multilobular nucleus and have a 12–15 µm diameter [2]. The
O’Sullivan and Yoshiro Kobayashi
different types of granules include (a) the primary/azurophilic, (b) secondary/specific and
Received: 27 September 2022 (c) tertiary, which differentiate them into three distinct categories. The primary granules
Accepted: 10 December 2022 contain myeloperoxidase (MPO), defensins, elastase and cathepsins, while the secondary
Published: 13 December 2022 granules contain NADPH oxidase, lactoferrin and gelatinase. Finally, the tertiary vesicles
consist of alkaline phosphatase and gelatinase [3,4].
Publisher’s Note: MDPI stays neutral
The traditional concept that neutrophils are terminally differentiated cells with limited
with regard to jurisdictional claims in
published maps and institutional affil-
plasticity and highly conserved function due to their low transcriptional activity has been
iations.
critically revised. Recent studies have provided evidence suggesting that neutrophils
express a wide variety of surface receptors that give them the ability to quickly respond
against disease–environmental cues and undergo transcriptional reprogramming, allowing
them to acquire disease-specific phenotypes [5,6]. Thus, neutrophils are considered a cell
Copyright: © 2022 by the authors. population with diverse functions, plasticity and a longer survival time of a few days than
Licensee MDPI, Basel, Switzerland. the one initially suggested of 8–10 h [7].
This article is an open access article High-throughput approaches have revealed that neutrophil gene expression includes
distributed under the terms and inflammatory agents, such as cytokines and chemokines, that complement the compo-
conditions of the Creative Commons nents [8,9] and factors involved in inflammation resolution [10]. Neutrophils interact with
Attribution (CC BY) license (https:// a variety of cell populations during their migration toward tissues, including endothelial
creativecommons.org/licenses/by/ cells, fibroblasts, macrophages, lymphocytes and dendritic cells [2].
4.0/).

Int. J. Mol. Sci. 2022, 23, 15823. https://doi.org/10.3390/ijms232415823 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 2 of 17

Int. J. Mol. Sci. 2022, 23, 15823 2 of 16

In the early 2000s, advances in molecular biology proposed the most important up-
date of Inneutrophil physiology,
the early 2000s, advancesparticularly
in moleculartheir capacity
biology to release
proposed the most neutrophil
importantextracellu-
update
lar traps (NETs) to kill bacteria [11]. Activated neutrophils generate
of neutrophil physiology, particularly their capacity to release neutrophil extracellular NETs, whichtraps
are ex-
tracellular
(NETs) to kill bacteria [11]. Activated neutrophils generate NETs, which are extracellular re-
fibers composed of chromatin, histones and granular proteins [11]. A NET
lease
fibersiscomposed
a mechanism through which
of chromatin, a neutrophil
histones and granularreleases NETs
proteins andAeventually
[11]. NET release dies
is [12];
a
however,
mechanism a NET
throughrelease
which is anot always linked
neutrophil releasestoNETs
the death of the neutrophil
and eventually [12]. Overall,
dies [12]; however, a
there
NET are three
release different
is not alwaystypes
linked oftoNETs (Figure
the death 1).neutrophil
of the In the first[12].
type, NETs there
Overall, are composed
are three of
different typesDNA
mitochondrial of NETs (Figure
together 1). In
with the firstproteins,
granular type, NETs andare composed of
neutrophilic mitochondrial
death does not fol-
DNA
low together
(Figure with granular
1) [13]. The otherproteins,
two typesandofneutrophilic death does
NETs are called vitalnot
andfollow (Figure
suicidal. The1)suicidal
[13].
The other two types of NETs are called vital and suicidal.
mechanism of NETs is dependent on the NADPH oxidase-2 enzyme and is accompanied The suicidal mechanism of
NETs is dependent on the NADPH oxidase-2 enzyme and is accompanied
by the death of neutrophils (Figure 1) [12]. The activation of NADPH oxidase relies on an by the death
of neutrophils
increase in the Ca (Figure 1) [12]. Theinactivation
2+ concentration of NADPH
the cytoplasm oxidase relies
and sometimes on on
thean increase of
generation
in the Ca2+ concentration in the cytoplasm and sometimes on the generation of ROS in
ROS in mitochondria [14]. On the other hand, vital NETs are independent of NADPH
mitochondria [14]. On the other hand, vital NETs are independent of NADPH oxidase-2,
oxidase-2, and neutrophils remain alive (Figure 1) [14]. A NET release can be induced in
and neutrophils remain alive (Figure 1) [14]. A NET release can be induced in vitro by
vitro by either inflammatory molecules (i.e., cytokines, pathogen components) that simu-
either inflammatory molecules (i.e., cytokines, pathogen components) that simulate the
late the disease
disease microenvironment
microenvironment or chemicalor chemical
compounds compounds
(e.g., PMA,(e.g., PMA, ionomycin)
ionomycin) that activatethat
activate
some intracellular pathways [15–17]. Each stimulus may induce a different different
some intracellular pathways [15–17]. Each stimulus may induce a pathway path-
of
way of NETs (Figure 1) [12]. The morphology of NETs, depending
NETs (Figure 1) [12]. The morphology of NETs, depending on the available space, is eitheron the available space,
iswide
eitheror wide or cloud-like
cloud-like [15,18,19].[15,18,19].

Figure 1. Schematic diagram demonstrating the different types of NETs. Depending on the stim-
ulation,
Figure 1. various molecular
Schematic diagram pathways result inthe
demonstrating thedifferent
generation
typesof NETs. ThisDepending
of NETs. diagram presents
on the the
stimu-
lation, variouspathways
fundamental molecularofpathways result inand
NET production, the in
generation
particular,ofthe
NETs.
vitalThis
NETsdiagram
or NADPHpresents the fun-
oxidase-2
damental pathways of
(NOX) -independent NET production,
pathway andNETs
and the suicidal in particular,
or NADPH the vital NETs
oxidase-2 (NOX)or -dependent
NADPH oxidase-2
path-
(NOX) -independent
way. Furthermore, pathway
NETs and
can also the suicidal
consist NETs or NADPH
of mitochondrial DNA (mtDNA) oxidase-2 (NOX)
rather than-dependent
chromosomal path-
way. Furthermore, NETs can also consist of mitochondrial DNA (mtDNA) rather than
DNA. In this pathway, intracellular grey structures represent mitochondrial organelles. Some indica-chromosomal
DNA. In this
tive NET pathway,
inducers intracellular
are chemical grey structures
compounds represent
or inflammatory mitochondrial
agents organelles. Some indic-
(Phorbol-12-myristate-13-acetate
ative NET inducers are chemical compounds or inflammatory agents (Phorbol-12-myristate-13-ac-
(PMA), antibodies (Abs), lipopolysaccharides (LPS), activated platelets, granulocyte-macrophage
etate (PMA), antibodies
colony-stimulating factor(Abs), lipopolysaccharides
(GM-CSF) (LPS), activated
and complement component C5a). platelets, granulocyte-macro-
phage colony-stimulating factor (GM-CSF) and complement component C5a).
Once neutrophils are activated, several downstream cellular events occur. During NET
Once neutrophils
formation, are activated,
neutrophil elastase several downstream
(NE), peptidyl-arginine cellular
deaminase events
4 (PAD4) andoccur. During
myeloper-
NET formation,
oxidase neutrophil
(MPO) migrate to the elastase
nucleus. (NE),
Indeed,peptidyl-arginine deaminase
PAD4 binds with calcium, 4 (PAD4)
and the complexand
is then transferred(MPO)
myeloperoxidase to the nucleus,
migrate where
to the histone
nucleus.hypercitrullination
Indeed, PAD4 binds and DNA
with deconden-
calcium, and
the complex is then transferred to the nucleus, where histone hypercitrullination and
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 3 of 17
Int. J. Mol. Sci. 2022, 23, 15823 3 of 16

DNA are
sation decondensation
mediated [14].areThe
mediated [14]. The
cytoskeletal cytoskeletal
components andcomponents and nucleus
nucleus envelope, as wellenve-
lope, as well as chromatin, are disintegrated by serine proteases. Finally, DNA,
as chromatin, are disintegrated by serine proteases. Finally, DNA, citrullinated histones citrulli-
nated histones
(CitH3) (CitH3)
and granular and are
proteins granular proteins
released into the are released environment
extracellular into the extracellular environ-
through pores
ment
on the through pores on the
cellular membrane cellular
(Figure 2). membrane (Figure 2).

Figure2.2. Immunofluorescence
Figure Immunofluorescencestaining:staining:AAqualitative
qualitativetool
toolfor
for NET
NET detection
detection inin human-isolated
human-isolated neu-
trophils using either a confocal or a conventional fluorescence microscope. Neutrophils
neutrophils using either a confocal or a conventional fluorescence microscope. Neutrophils isolated isolated
from healthy donors’ whole blood using a density gradient separation method were
from healthy donors’ whole blood using a density gradient separation method were stimulated with
stimulated with
a serum derived from a treatment-naïve patient with active ulcerative colitis (180 min incubation
a serum derived from a treatment-naïve patient with active ulcerative colitis (180 min incubation
time). NETs are visualized as fibers of extracellular DNA and neutrophil granule proteins assessed
time). NETs are visualized as fibers of extracellular DNA and neutrophil granule proteins assessed by
by double-staining with a chromatin-staining dye and a neutrophil marker. (A) Neutrophils were
double-staining with a chromatin-staining dye and a neutrophil marker. (A) Neutrophils were stained
stained with a rabbit anti-human neutrophil elastase ((NE), Abcam) antibody detected by a goat
with a rabbit anti-human neutrophil elastase ((NE), Abcam, Cambridge, UK) antibody detected by
anti-rabbit AlexaFluor 594 antibody (Invitrogen). Visualization was performed using confocal mi-
acroscopy
goat anti-rabbit AlexaFluor
(Revolution spinning 594disk
antibody (Invitrogen,
confocal Waltham,
system; Andor, MA, USA).
Ireland). Visualization
(B) Neutrophils was
were stained
performed using confocal microscopy (Revolution spinning disk confocal system; Andor,
with a mouse anti-human neutrophil elastase ((NE), Abcam) antibody detected by a goat anti-mouse Belfast,
UK). (B) Neutrophils
AlexaFluor were stained
488 antibody with aVisualization
(Invitrogen). mouse anti-human neutrophil using
was performed elastase ((NE), Abcam)
a fluorescence micro-
antibody detected by a goat anti-mouse AlexaFluor 488 antibody (Invitrogen). Visualization
scope (OLYMPUS BX51). In (A,B), cell nuclei were counterstained with 4′,6-diamidino-2-phenylin- was
performed usingSigma-Aldrich).
dole ((DAPI), a fluorescence microscope
Untreated(OLYMPUS
neutrophilsBX51, Shinjuku,
served Japan).Data
as control. In (A,B),
fromcell
thenuclei
Molecular
0
Hematology
were Laboratory
counterstained with 4archive are shown.
,6-diamidino-2-phenylindole ((DAPI), Sigma-Aldrich, Burlington, MA,
USA). Untreated neutrophils served as control. Data from the Molecular Hematology Laboratory
archiveSince NET formation was first reported in 2004 as an eliminating mechanism for in-
are shown.
vading microbes [11], NETs have attracted much interest, and several of their additional
Since NET formation was first reported in 2004 as an eliminating mechanism for
functions have been discovered. Another important role of NETs is the activation of other
invading microbes [11], NETs have attracted much interest, and several of their additional
immune cells during inflammation, either to orchestrate an inflammatory response or to
functions have been discovered. Another important role of NETs is the activation of other
resolve inflammation [20]. Aggregated NETs contribute to degrading cytokines and
immune cells during inflammation, either to orchestrate an inflammatory response or
chemokines,
to resulting in
resolve inflammation inflammation
[20]. Aggregatedresolution in the acute
NETs contribute inflammatory
to degrading response
cytokines and of
gout [21].
Accumulating evidence suggests that NETs induced by different stimuli and disease
conditions may be heterogeneous in regard to protein composition and post-translational
modifications acquiring different biological effects [16,22]. In addition to DNA and
Int. J. Mol. Sci. 2022, 23, 15823 4 of 16

chemokines, resulting in inflammation resolution in the acute inflammatory response of

gout [21].
Accumulating evidence suggests that NETs induced by different stimuli and disease
conditions may be heterogeneous in regard to protein composition and post-translational
modifications acquiring different biological effects [16,22]. In addition to DNA and granular
proteins, NETs contain disease-related proteins that have a specific role in numerous human
disorders. In particular, NETs have been proposed to induce the formation of autoantibod-
ies in diseases, such as small vessel vasculitis and systemic lupus erythematosus (SLE) [23],
as well as acting as functional scaffolds for thrombosis by aggregating platelets [24]. Fur-
thermore, NETs participate in chronic inflammation [25] and fibrotic disorders via cross-talk
between NETs and mesenchymal cells [26].
Because of this vital clinical significance, the role of neutrophils/NETs as a pivotal
therapeutic target in inflammatory pathology has been reconsidered. Therefore, either the
spontaneous formation of NETs (ex vivo biological sample) or the in vitro-induced NETs
should be easily and efficiently studied by the application of the appropriate laboratory
methods. Here, we present and discuss the updated tools available for quantitative and
qualitative evaluation of this mechanism.

2. Tools for the Assessment of NETs

2.1. Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA is a commonly used tool in NET research, allowing NET detection and quan-
tification. This method provides clinical significance by associating NET markers with
human disorders, such as cancer [27], autoimmune small-vessel vasculitis [28,29], rheuma-
toid arthritis [30], ulcerative colitis [31], lupus [32–34], SARS-CoV-2 infection [35,36] pul-
monary [37,38] and cystic fibrosis [39,40]. There are several validated, commercially avail-
able ELISA tests for detecting NETs [41], as well as in-house sandwich ELISA assays [42].
The scientific community is still in controversy regarding which is the most targeted
biomarker for detecting NET formation with the ELISA technique. In particular, detecting
nucleosomes and circulating cell-free DNA does not always indicate NETs since they may
be derived from other events, such as apoptosis or necrosis [43]. Similarly, several ELISA
kits have neutrophil-derived enzymes, such as myeloperoxidase and neutrophil elastase,
as NET markers that may not accurately reflect neutrophil activation, degranulation and
NET formation [44].
In 2017, an ELISA was presented for detecting citrullinated histone H3 (CitH3) in
human plasma. While the assay detects NET formation with high specificity, CitH3 as a
NET biomarker is specific only to the PAD4-dependent pathway [45]. Two years later, an
optimized nucleosomal CitH3-DNA ELISA was validated for reliable NET quantification
in human plasma [46]. Complexes of extracellular DNA with myeloperoxidase (MPO) [29]
and neutrophil elastase (NE) [44] are considered specific NET biomarkers. Methods that
detect the presence of two markers are better. However, a recent study alerted the NET
community about the specificity of MPO-DNA complexes with ELISA [47]. An MPO-DNA
ELISA proved to be specific for NET formation in vitro but was unspecific to in vivo and no
correlation between plasma MPO-DNA complexes and other NET biomarkers were found,
suggesting the need to use isotype control antibodies and additional NET markers [47].
The significant advantage of NET quantification by ELISA is the availability of several
ELISA kits, as it is easy to use [41]. Furthermore, NET detection by ELISA is an uncompli-
cated procedure with high specificity and accessible equipment by several laboratories [48].
Compared to other techniques applied in NET research, ELISA assays have a low cost
(Table 1) [41]. Nevertheless, NET quantification by ELISA requires assay standardization,
as several employable ELISA kits offer limited reproducibility of the results [46]. An addi-
tional challenge is the evidence of a restricted correlation between detecting NET formation
by ELISA in vitro and in vivo (Table 1) [46,47].
Int. J. Mol. Sci. 2022, 23, 15823 5 of 16

Table 1. The benefits and the limitations of each laboratory method used in the context of NET evaluation.

Techniques Benefits Limitations References

Enzyme-linked Simplicity, specificity, Results standardization and Kasprzycka et al., 2019 [41];
immunosorbent cost-effectiveness, differences between in vivo Thålin et al., 2017 [45];
assay (ELISA) objective and quantitative and in vitro NETs Masuda et al., 2017 [49]
A time-consuming procedure, Brinkmann et al., 2004 [11];
Distinguishes cell death laborious, limited Von Köckritz-Blickwede et al.,
mechanisms, objective reproducibility, not available 2010 [50];
Immunofluorescence quantification of NETs with for rapid screening of many Brinkmann et al., 2012 [51];
microscopy (IFM) software-based methods, and cells or samples, Coelho et al., 2015 [52];
detects NETs in vitro, ex vivo observer-dependent, and De Buhr and von
and in situ extensive precaution with Köckritz-Blickwede, 2016 [15];
sample preparation Lv et al., 2020 [53]
Fuchs et al., 2007 [24];
Biased based on the field of
Distinguishes cell death De Buhr and von
view and no reliable
Electron microscopy mechanisms and detects NETs Köckritz-Blickwede, 2016 [15];
distinguishing between NETs
in vitro and in situ Krautgartner et al., 2008 [54];
and fibrin by SEM
Lv et al., 2020 [53]
Live in vivo imaging using
animal models, close Staining of many components Cho et al., 2011 [55];
Live imaging examination of NET structures, on NETs, complex equipment Kolaczkowska et al., 2015 [56];
and distinguishes NETs from and expensive Gupta et al., 2018 [57];
other cell deaths
Qualitative, quantitative and
objective; detects Extensive precaution with
Masuda et al., 2017 [49];
pre-NETotic cells, both in vivo sample preparation; detects
Kasprzycka et al., 2019 [41];
Flow cytometry and in vitro, rapid and sort only pre-NETotic cells and is
Gavillet et al., 2015 [58];
between cell populations and incapable of detecting
Zharkova et al., 2019 [59]
has the possibility of direct released NETs/remnants
applications in blood samples
Rapid, semiautomated and
Sophisticated equipment and
Multispectral image flow studies subcellular morphology
detects fewer NETotic cells Zhao et al., 2015 [60]
cytometer (MIFC) and distinguishes cell
than existing
death mechanisms
Semi-quantitative, Multi-step protocol and
Liu et al., 2016 [61];
Western blot specificity and optimizes the concentration of
Lv et al., 2020 [53]
sensitivity the primary antibody

2.2. Immunofluorescence Microscopy

NET visualization by immunofluorescence microscopy is a commonly applied methodol-
ogy based on immunostaining of NET components, such as granular staining for neutrophil-
associated enzymes and nuclear staining for histones and extracellular DNA, allowing
the visualization of their expression (Figures 2 and 3) [11]. The basic strategy of this
methodology to detect NETs is using DNA intercalating dyes to visualize DNA and specific
immunostaining for the enzymatic components of NETs to improve quantification and visu-
alization (Figures 2 and 3) [15]. Using extracellular DNA and neutrophil-derived proteins
(e.g., CitH3, MPO, NE etc.), a NET formation can be detected by immunofluorescence mi-
croscopy in several biological specimens, such as tissue sections, peripheral blood samples,
sera, bronchoalveolar lavage fluids and cell culture mediums (Figures 2 and 3) [53]. Im-
munofluorescence microscopy can characterize NETs in vitro as a response to pathogens or
chemical agents in tissue culture plates, ex vivo in blood samples and in situ in histological
samples from humans or animals [15].
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 6 of 17

(Figures 2 and 3) [53]. Immunofluorescence microscopy can characterize NETs in vitro as

Int. J. Mol. Sci. 2022, 23, 15823 6 of 16
a response to pathogens or chemical agents in tissue culture plates, ex vivo in blood sam-
ples and in situ in histological samples from humans or animals [15].

Figure3.3.Immunofluorescence
Figure Immunofluorescence staining:
staining: a qualitative
a qualitative tooltool to detect
to detect NETNET remnants
remnants or disease-related
or disease-related
proteins located on NETs in human tissue specimens. Cross-sections (4 μm
proteins located on NETs in human tissue specimens. Cross-sections (4 µm thickness) of kidneythickness) of kidney
biopsy tissue derived from a patient with proliferative lupus nephritis (LN) were
biopsy tissue derived from a patient with proliferative lupus nephritis (LN) were deparaffinized deparaffinized
and stained with the appropriate antibodies. (A) NETs were visualized in kidney specimens as ex-
and stained with the appropriate antibodies. (A) NETs were visualized in kidney specimens as
tracellular structures by staining with both a mouse anti-human neutrophil elastase ((NE), Abcam)
extracellular structures by staining with both a mouse anti-human neutrophil elastase ((NE), Abcam)
antibody and a rabbit anti-human histone 3 (Anti-Histone H3 (citrulline R2 + R8 + R17), Abcam)
antibody and a rabbit anti-human histone 3 (Anti-Histone H3 (citrulline R2 + R8 + R17), Abcam)
antibody. (B) The presence of a disease-related protein on extracellular structures, such as IL-17 in
antibody. (B) The presence of a disease-related protein on extracellular structures, such as IL-17 in
LN, was examined using both a mouse anti-human IL-17A (R&D Systems) antibody and a rabbit
LN, was examined
anti-human using
histone H3 both a mouse anti-human
(Anti-Histone IL-17A
H3 (citrulline R2 +(R&D
R8 + Systems) antibody
R17), Abcam) and a rabbit
antibody. In (A,B), a
anti-human histone H3 (Anti-Histone H3 (citrulline R2 + R8 + R17), Abcam)
goat anti-mouse AlexaFluor 488 antibody (Invitrogen) and a goat anti-rabbit AlexaFluor antibody. In (A,B),
594a anti-
goat
bodyanti-mouse
(Invitrogen) AlexaFluor 488 to
were used antibody (Invitrogen)
detect primary and a goat
antibodies. anti-rabbit
Sections wereAlexaFluor 594 antibody
counterstained with DAPI
(Invitrogen)
and visualizedwereinused to detect
a confocal primary antibodies.
microscope (Revolution Sections were
spinning counterstained
disk with DAPI
confocal system; and
Andor, Belfast,
visualized in a confocal
Ireland). Data from themicroscope
Molecular(Revolution
Hematology spinning disk confocal
Laboratory archive system; Andor, Belfast, UK).
are shown.
Data from the Molecular Hematology Laboratory archive are shown.
Int. J. Mol. Sci. 2022, 23, 15823 7 of 16

A great benefit of detecting NETs by immunofluorescence microscopy is the visible

differentiation of the distinct stages of NETs based on the morphological changes of the
nucleus [50]. Furthermore, immunofluorescence microscopy allows visible differentiation
between NETs apoptosis and necrosis [50]. Immunofluorescence staining of virulence
factors enables the visualization of them entrapped in NETs [11]. Another strength of this
technique is that specimens treated with immunostaining can be stored for up to six months
at 4 ◦ C (Table 1) [50].
However, detecting NET release by immunofluorescence microscopy can be observer-
dependent based on the field of view [15]. Apart from being laborious and time-consuming,
the quantification of NET release is prone to errors with limited data reproducibility due to
subjectivity [52]. Another drawback is the intensive precaution during sample fixing and
staining [62]. Furthermore, this technique is suitable only for analyzing a small number of
cells in a sample (Table 1) [15].
As mentioned in the Introduction section, NETs are also enriched with a variety of
disease-related proteins closely related to the pathophysiology of each disorder (“disease-
related” proteins) [16]. For instance, bioactive tissue factor (TF) was detected on NETs
in vein and arterial thrombosis [24,63] and mature interleukin-1 beta (IL-1β) in Familial
Mediterranean Fever [64] and gouty arthritis [65]. Hence, in a disease model, the expression
of “disease-related” proteins on the NET scaffold can be studied by applying specific
antibodies that target them. In order to concomitantly verify that these proteins are derived
from neutrophils that are undergoing NET formation, specimens could be co-stained with
robust markers of NETs, including at least two antibodies against neutrophil-derived
proteins (CitH3, NE or MPO) and DNA intercalating dyes (Figure 3) [51,66,67].
A selected example for the assessment of NET formation by immunofluorescence
microscopy is the study of Brinkmann et al., who validated a semi-automatic protocol
for NET quantification in vitro. Specifically, it involved using antibodies against a subnu-
cleosomal complex (histone 2A, histone 2b and chromatin) and neutrophil elastase. The
number of cells per field was determined by staining cells with the DNA-intercalating dye,
Hoechst 33342. This method is based on the observation that anti-chromatin antibodies
can more easily and directly bind to decondensed chromatin, characterizing the cells that
undergo NETs. A correlation was identified between the fluorescence signals of the anti-
chromatin antibody and the signals of DNA-binding dye, resulting in the calculation of
the percentage of netting neutrophils [51]. Data interpretation of this technique does not
require complex equipment, only a fluorescence microscope and a public-domain software
package [51]. Furthermore, Coelho et al. used an antibody against the complex of histone-
DNA and DAPI for DNA staining, detecting NETs with immunofluorescence microscopy
and open-source software [52]. Similarly, von Köckritz-Blickwede et al. used an antibody
against the complex of histones-DNA and DAPI, detecting NETs with immunofluorescence
microscopy; however, they examined the samples only with the aid of a microscope without
using software [50]. Notably, the quantification of NETs with software-based methods is
unbiased, objective and valuable for data standardization and comparison [51].
At the time of this writing, the 3D9 monoclonal antibody against the cleavage site of
histone H3 (H3R49) has been reported as a novel tool for detecting and quantifying NET
formations in humans by using immunofluorescence microscopy in purified neutrophils
and tissue sections [68]. Although ex vivo validation of this method in serum/plasma
is pending, it appears to be a specific marker of NETs and distinguishes NETs from the
chromatin of other cell fractions and neutrophils that die via alternative mechanisms, such
as apoptosis, necrosis and necroptosis [68].

2.3. Electron Microscopy

Another method for NET visualization is electron microscopy. It is a quite common
and relatively old technique based on the acceleration of electrons to visualize a sample
placed in a vacuum chamber [69]. Both transmission electron microscopy (TEM) and
scanning electron microscopy (SEM) methods are based on an electron beam, either passing
Int. J. Mol. Sci. 2022, 23, 15823 8 of 16

through or reflecting from the surface of the sample. In TEM, the electron beam passes
through the sample and is projected onto a fluorescent screen, producing an image [70]. In
contrast, in SEM, the electron beam reflects upon one area of the sample and the emitted
signal is then collected by detectors where an image is produced [54,70]. Additionally,
TEM is mainly used for visualizing the inner parts of a sample, whereas SEM is used for
the surface examination of a sample [54]. TEM provides a higher image resolution with
the image projected in 2D, while SEM yields a greater depth of field, thus producing a 3D
image [54,70].
Electron microscopy is a beneficial methodology to assess NETs (Table 1). According to
the literature, SEM seems to be the central choice for visualizing NET formation. In general,
the electron microscope provides a better image resolution and magnification compared to
conventional light microscope visualization [54]. Thus, NETs can be differentiated from
fibrins when high-resolution SEM is applied [20]. Nevertheless, the high energy projected
from the electron beam can sometimes change the structure of a sample and therefore
provide a faulty image. That is why special preparation of the sample is required, which
can often be a very complex and expensive procedure (Table 1) [54].
Conventional electron microscopy staining methods still cannot visualize NET forma-
tion as accurately. There is a reliable protocol where TEM can also be used to demonstrate
NETs and interactions with bacteria [71]. Nevertheless, TEM is a distinctive visualization
method for autophagy [71], as it allows the visualization of the inner parts of a cell. Au-
tophagy, which is closely associated with the release of NETs [18,72], is an intracellular
mechanism and can therefore be visualized in great depth. The work of Tang et al. demon-
strated the increased vacuolization that neutrophils undergo when treated with H4B4 (an
anti-human LAMP-2 antibody), which in turn led to an increase in NETs [73]. Electron
microscopy was also used in the work of Fuchs et al., where an interesting connection
between NETs and platelets was demonstrated where NETs played a promoting role to-
ward thrombosis [24]. In addition, Ermert et al. used SEM to observe NET formation in
neutrophils isolated from mice, while Manzenreiter et al. investigated the complex role
of NETs in cystic fibrosis by SEM [74,75]. In more recent work, Rajeeve et al. investigated
NET formation through the infection of neutrophils with Chlamydia trachomatis, which
normally blocks NET production, but when the chlamydia protease CPAF is absent from
the bacteria, NETs are observed [55].

2.4. Live Imaging

Intravital microscopy allows imaging of immune responses at a molecular level while
the animal is alive, usually involving either confocal or multiphoton microscopy [76]. The
basis of multiphoton microscopy is photon-induced fluorescence excitation and subsequent
electron emission detection. It has several advantages compared with confocal microscopy,
as it allows deeper tissue penetration and involves less phototoxicity [76]. In contrast
to confocal microscopy, it does not require a pinhole to reject out-of-focus light but has
inherited optical capability [76,77]. However, this is solved with the addition of the spinning
disk to the confocal microscope, which allows the generation of multiple excitation points
at the same time and not just one, leading to reduced phototoxicity [56,78].
Specifically, regarding NETs, it allows real-time NET visualization. The basic principle
of this methodology is staining components that indicate NET release to identify their
location and visualize their structure [79]. Furthermore, intravital imaging enables the close
examination of the NET structure (Table 1). However, the application of this technique
to identify NETs in tissue specimens may demand the staining of several distinct compo-
nents of NETs due to overlapping antibodies and signal emissions from different cellular
components (Table 1) [79].
Kang et al. applied this technique to visualize NET formation with the detection
of extracellular DNA using Sytox Green in the peri-infarct cortex of mice and associated
NETs as toxic signals for the remodeling process that occurs after a stroke [80]. Another
study involved the use of spinning disk confocal intravital microscopy to visualize NETs
Int. J. Mol. Sci. 2022, 23, 15823 9 of 16

by staining neutrophil elastase (NE) and Ly6G in obese mice, where NETs are impaired due
to obesity because of the presence of dysfunctional platelets [81], which have been shown
to play an important role in sepsis [82]. This study aimed to gain further insights regarding
the obesity paradox in sepsis, showing reduced inflammation in the liver of obese mice [81].
Hoppenbrouwers et al. also used this technique in an effort to assess the robustness
of different NET inducers when stimulating neutrophils from healthy individuals with
different molecules in vitro [57], including the organic molecule phorbol 12-myristate
13-acetate (PMA), ionomycin, lipopolysaccharide (LPS) and gram-positive or -negative
bacteria [57]. Their study has suggested PMA, ionomycin and bacteria as strong inducers
of NETs. Their use of time-lapse imaging also revealed a different series of events when
comparing the NET process triggered by ionomycin and PMA [57].
A novel method for a quick, high-throughput and reproducible quantification of NET
release in real-time is the use of the IncuCyte ZOOM Imaging Platform [83]. This method
can identify NETs from other cell death pathways by detecting morphological changes
in cells, nuclei and kinetics [83]. Since the IncuCyte ZOOM Imaging Platform permits
the use of only two different fluorescent signals, it limits the ability to detect additional
neutrophil components. Overall, this platform uses three imaging channels: the red
fluorescent channel, the green fluorescent channel and the phase contrast channel [83]. The
requirement for complex equipment and the potential for false-positive results when the
plasma membrane integrity is compromised are additional drawbacks of this technique [83].

2.5. Flow Cytometry

Flow cytometry is an attractive tool for evaluating NETs that characterizes single-cell
morphology, size and granularity [58]. The basic principle of flow cytometry relies on
light scattering and fluorescence emission from dyes or conjugated antibodies, which bind
to cytokines, receptors, cellular components and DNA, leading to the differentiation of
distinct cell populations in whole blood (Figure 4) [58].
Some well-established protocols in NET research include staining with fluorescent
dyes or monoclonal antibodies specific to cell surface markers or intracellular markers of
NETs. Key components of NETs could be detected using flow cytometry with fluorescently
labeled antibodies together with DNA dyes indicating NETs (Figure 4) [49].
Gavillet et al. developed a flow cytometry protocol, detecting MPO and citrullinated
histones in vivo, directly in whole blood samples, without the need to isolate neutrophils.
Thus, this flow method allows rapid assessment of NETs and quantification in a large
cell population [49]. This methodology applied the detection of netting neutrophils both
in vivo in clinical blood samples and in in vitro conditions [49]. In 2017, Masuda et al.
proposed a quantitative, simple Flow Cytometry-assay that detected NETs by using a DNA-
binding dye (Sytox Green), but it was unable to pass through the membrane, indicating
that green-stained cells underwent NETs [59]. Zharkova et al. suggested the application of
a fluorescently labeled antibody mixture against neutrophil cell surface markers [84]. This
allows the direct detection of NETs in a mixed population.
Flow cytometry allows rapid screening of a large population of neutrophils in a small
sample volume and provides detailed qualitative and quantitative information with high
efficiency in a short time [41]. Additionally, this methodology allows NET quantification
with high objectivity [85]. Hence, this technique is a notable diagnostic tool, associating NET
generation with several human disorders (Table 1) [85]. However, the reproducibility of
the results requires extensive precaution during sample preparation [84], which is the main
drawback of this technique. Given that flow cytometry detects intact cells, this technique
allows the detection of NETotic potential during the early stages, i.e., pro-NETotic cells,
before they die by forming NETs (Table 1).
Int. J. Mol. Sci. 2022, 23, 15823 10 of 16
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 10 of 17

Figure
Figure 4. Flow cytometry 4. Flowan
assay: cytometry assay:
indicative an indicative quantitative
quantitative method ofmethod of NET determination
NET determination in human
in human
isolated neutrophils. Flow cytometric analysis of human neutrophils derived from a healthy donor.
isolated neutrophils. Flow cytometric analysis of human neutrophils derived from a healthy donor.
Neutrophils were isolated from whole blood using a density gradient separation method. (A)
Neutrophils were isolated from whole blood using a density gradient separation method. (A) Unstim-
ulated neutrophils (negative control) or (B) neutrophils stimulated with a chemical inducer of NETs
(phorbol 12-myristate 13-acetate (PMA), Sigma-Aldrich; 20 nM final concentration, 45 min incubation
time) were stained with a neutrophil activation marker (mouse anti-human CD66b—conjugated
with PerCP/Cyanine5.5, Biolegend, San Diego, CA, USA). CD66b positive cells were examined for
the presence of citrullinated histone 3 upon staining with a rabbit anti-human histone 3 antibody
(Recombinant anti-histone H3 (citrulline R8), Abcam) detected with a goat anti-rabbit AlexaFluor488
antibody (Invitrogen). It was tested that PMA did not induce apoptosis/necrosis in neutrophils under
these experimental conditions. Data from the Molecular Hematology Laboratory archive are shown.
Int. J. Mol. Sci. 2022, 23, 15823 11 of 16

2.6. Multispectral Image Flow Cytometry (MIFC)

Multispectral Imaging Flow Cytometry (MIFC), based on immunofluorescence and
fluorescent-activated cell sorting (FACS), can semi-automatically quantify NET formation
in several samples [60]. In 2015, Zhao et al. made use of MIFC to measure both “suicidal”
and “vital” NETs, investigating the morphological changes of netting neutrophils [86].
Imaging of neutrophils in a fluid stream provides information about the nuclear changes,
allowing the characterization of the cell death mechanism since the formation of blebs can
be clearly distinguished [86]. In the study of Zhao et al., MPO was co-localized with DNA
in “suicidal” NETs.
Apart from distinguishing the cell death process (“suicidal” NETs, “vital” NETs and
apoptosis), MIFC adds several additional benefits to NET research. This technique provides
simplicity and reproducibility of quantitative results while being available for several
sample analyses [60]. Detecting NETs by MIFC automatically and rapidly might enable
the use of NET formation as a biomarker of human diseases and chemical compound
screening to investigate the reflection on the NET formation (Table 1) [86]. However, the
primary disadvantage of this technique is the capture of only netting neutrophils in the
early phase, which might underestimate neutrophils in later phases of this mechanism [86].
Furthermore, MIFC may miss neutrophils undergoing “vital” NETs [86], and this technique
requires sophisticated equipment for data acquisition (Table 1) [86].

2.7. Western Blot

Western blot or immunoblotting is a technique that can be used for NET detection.
In the same context, disease-specific proteins exposed on NETs could also be detected
by immunoblotting.
Similar to ELISA, it is a frequently used technique for antigen detection [87]. Specifically,
with Western blot, a protein or antigen can be detected among multiple proteins/antigens
in a host. The process, in brief, involves the separation of proteins based on the size in an
SDS-polyacrylamide gel and the subsequent transfer of these proteins to a nitrocellulose
membrane. A primary antibody specific to the protein of interest is added to this membrane.
Detection is performed either directly, meaning that the label will be on the primary
antibody, or indirectly, meaning that a secondary labeled antibody will be added [87].
The markers used to detect NETs are mainly extracellular and modified histones,
such as H2A/H2B or CitH3. PAD4 can also serve as a marker. Pulavendran et al. used
labeled antibodies against these specific markers to evaluate NETs in BAL fluids isolated
from mice infected with a strand of bacteria called Francisella tularensis, which is the cause
of pneumonic tularemia. The infected mice showed higher levels of NETs than healthy
mice [61]. Liu et al. also used CitH3 as a marker to assess NET formation with Western
blot in additional experiments of a phenomenon called ALI, or acute lung injury, mediated
by LPS. In particular, they isolated lung tissue from mice treated with LPS and noticed an
increase in CitH3. Therefore, NETs were present in the tissue of these mice rather than
in the control mice. These NETs could represent the source of organ damage and the
initial inflammatory reaction. Notably, the activation is not direct, as LPS alone cannot
efficiently induce NET formation, but platelets that are activated by LPS can indeed make
this happen [88]. Similarly, our group has also applied CitH3 as a protein marker during
the immunoblotting process to support that fibrosis-related agents are able to trigger
NET release in neutrophils obtained from healthy individuals. Indeed, our data further
suggested that NETs generated upon this treatment are involved in the ensuing fibrosis,
providing a link between NET formation and human fibrotic diseases [26].
Western blot is a particularly beneficial technique because it is sensitive and specific
and provides some quantitative results based on the intensity of the bands (Table 1).
However, there are some disadvantages, such as the significant limitation of the need for
a primary antibody; otherwise, the technique cannot be performed. The most common
problem is the antibody’s required concentration that is needed to detect the specific protein
efficiently and not have background staining. These antibodies are also not very cheap and
Int. J. Mol. Sci. 2022, 23, 15823 12 of 16

often lose their specificity when thrown into a mix of many proteins. Finally, the protocol is
not standard, and the optimization process can sometimes prove difficult since Western
blot is a very tender and multi-step technique (Table 1) [89].

2.8. DNA Dyes

A rapid and easy method to detect NETs in a plate assay employs impermeable DNA
dyes, such as Sytox Green. This assay’s principle relies on the fact that the cell membrane is
intact before NET formation. An impermeable DNA dye (e.g., Sytox Green) can be used to
quantify the released cell-free DNA, including the formation of NETs as they are present
in the extracellular space. A permeable DNA dye (e.g., pico-Orange) can also be used to
quantify the total DNA, including intact cells, in order to measure a ratio of NET/total
DNA. If they have different emission spectrums and different region binding, then they
can be used at the same time in the same well. That means that one would have to bind in
the major grooves and the other in minor grooves. Quantification of the binding requires a
fluorometer [60]. Yost et al. utilized Sytox Orange, an impermeable dye, to assess NETs,
where neutrophils isolated from adults and neonates were stimulated with LPS, and it was
shown that (particularly in adult samples) extracellular DNA was higher; hence, a higher
number of NETs was present. Of course, they still used microscopy techniques with both
an impermeable (Sytox Orange) and a permeable dye (Syto Green, not to be confused with
the impermeable Sytox Green DNA dye) to further verify the induction of NETs a marker
for NE was also used [90]. Another more recent work by Hopke et al. regarding neutrophil
swarming and NET release against C. albicans, Sytox Green was used to visualize the
swarming effect and NETs. By measuring the fluorescence intensity of Sytox Green and the
number of nuclei, they confirmed that NETs were released after swarming the fungi, which
is an event that corresponds to the almost simultaneous recruitment of neutrophils against
a pathogen [91].
The main advantage of this technique is that it is quick and easy, where quantification
of DNA release and, therefore, NETs are available. On the other hand, it should not
be used as the only method to quantify NETs and is also not very reliable since any
effect/stimuli that disturb the integrity of the cell membrane will give false-positive results.
Therefore, distinguishing between NETs and necrosis or apoptosis is not possible unless
more markers are used [60]. In other words, cell-free DNA could originate from cells
other than neutrophils, and hence, there is a need to engage further in an ELISA assay
able to concomitantly measure extracellular DNA and a neutrophilic marker, such as
myeloperoxidase (MPO) or neutrophil elastase (NE) [26].

3. Concluding Remarks
The increasing implications of NET formation in neutrophil biology and translational
medical research require reliable and efficient tools for NET identification. Immunoassays,
such as ELISA and Western blot, are used for NET quantification, but the results require
standardization (Table 1). ELISA is a widely available technique due to its low cost, in-
creased accessibility and simplicity. Western blot can have high costs and be laborious
because of the extensive sample preparation. Flow cytometry-based approaches allow
NET quantification with high objectivity and reproducibility in a rapid manner. However,
sample preparation can be challenging. In addition, flow cytometry identifies and ana-
lyzes pro-NETotic cells prone to release NETs (Table 1). Visualization methods effectively
distinguish the cell death mechanism. Nevertheless, immunofluorescence and electron
microscopy are highly dependent on the observer and the field of view (Table 1). Therefore,
it seems that there is no gold-standard method for the assessment of NET formation. Until
today, a combination of different molecular techniques should be used to avoid possi-
ble misinterpretation of the results and provide the utmost comparable data. Moreover,
functional assays are used to demonstrate a definite biological role for NETs and various
NET-located proteins that are detected in the circulation and/or tissues. Considerably, the
Int. J. Mol. Sci. 2022, 23, 15823 13 of 16

clinical significance of accurate NET detection and the application of molecular biology
techniques in this field needs to be standardized and optimized further.

Author Contributions: M.S., G.T., P.S. and A.C. wrote the manuscript and created the Table/Figures.
P.S. and A.C. revised the manuscript. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We would like to gratefully acknowledge Konstantinos Ritis for his support and
advice in studying the mechanism of NETs in distinct inflammatory and thrombotic disorders. He
always acts as a constant source of inspiration for us. We also wish to thank all new and previous
members of the Laboratory of Molecular Hematology (Democritus University of Thrace, DUTH)
for their contribution to the immunofluorescence data presented in this review article. Finally, we
kindly acknowledge Maria Koffa for her guidance in CIBIT—Bioimaging Facilities of the Democritus
University of Thrace.
Conflicts of Interest: The authors declare no conflict of interest.

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