www.elsevier.com/locate/micron

Micron 39 (2008) 367–372

Visualization of neutrophil extracellular traps in TEM

Wolf Dietrich Krautgartner a, Ljubomir Vitkov b,c,*a Department of Light & Electron Microscopy, Organismic Biology, University of Salzburg, Hellbrunnerstraße 34, A-5020 Salzburg, Austria

b Department of Operative Dentistry & Periodontology, Medical University of Innsbruck, Anichstraße 35, A-6020 Innsbruck, Austriac Mayburgerplatz 7, A-5204 Straßwalchen, Austria

Received 25 February 2007; received in revised form 18 March 2007; accepted 19 March 2007

Abstract

Neutrophil extracellular traps (NETs) have recently been described as an important innate defence mechanism in inflammation. However,

routine electron microscopic staining techniques faintly stain NETs and are therefore insufficient for enabling a distinction between these and the

host cell debris as well as proteins regularly present at the site of inflammation. In order to test suitable electron microscopic staining techniques,

NETs induced ex vivo via phorbol myristate were absorbed on formvar. Four types of drop-on-grid positive staining were used: osmium tetroxide

(Os), osmium tetroxide–uranyl acetate–lead citrate (Os–U–Pb), ruthenium red-osmium tetroxide (RR-OsO4), and cuprolinic blue enhanced by

sodium tungstate (CB-WO4). We observed no staining of NETs using Os, faint staining with Os–U–Pb, better but still weak staining with CB-WO4

and outstanding staining with RR-OsO4. Furthermore, RR-OsO4 staining also enables the observation of bacterial fimbriae-mediated adhesion,

which is possibly responsible for the ability of NETs to bind bacteria. Thus, the offered RR-OsO4 staining technique may facilitate the study of the

NETs-bacterial interactions.

# 2007 Elsevier Ltd. All rights reserved.

Keywords: Cationic dyes; Cuprolinic blue; Ruthenium red; Deoxyribonuclease; Globular domains

1. Introduction

Recently, a ‘‘new’’ mechanism for bacterial clearance by

neutrophil extracellular traps (NETs) has been reported

(Brinkmann et al., 2004). NETs are extracellular net-like

fibres generated by activated neutrophils and are able to disarm

and kill bacteria extracellularly. They consist of a DNA

backbone with embedded antimicrobial peptides and enzymes,

e.g. histones and neutrophil elastase (Brinkmann et al., 2004;

Fuchs et al., 2007). NETs bind Gram-positive as well as Gram-

negative bacteria and are abundant in vivo in acute inflamma-

tion. They appear to be a form of innate response that binds

microorganisms, prevents them from spreading and ensures a

high local concentration of antimicrobial agents to degrade

virulence factors and kill bacteria (Brinkmann et al., 2004).

Although NETs are of pronounced interest to the

investigation of the host response to bacterial challenge, their

visualization in inflamed tissues by routine transmission

electron microscopy techniques is not reliable, as there is

* Corresponding author at: Mayburgerplatz 7, A-5204 Straßwalchen, Austria.

Tel.: +43 676 4041225; fax: +43 6215 20088.

E-mail address: lvitkov@yahoo.com (L. Vitkov).

0968-4328/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.micron.2007.03.007

insufficient staining when applying routine techniques. Thus,

the basic elements of NETs, the fibrils with a diameter of 15–

17 nm have not been visualized by transmission electron

microscopy (TEM) until now, but only by high resolution

scanning electron microscopy (Brinkmann et al., 2004; Fuchs

et al., 2007). Consequently, the question arises, whether a

selective staining for chromatin could enable the visualization

and differentiation of NETs by TEM in the presence of

extracellular host proteins regularly present at the site of

inflammation.

The aim of the present work was to offer a reliable method

for visualization and differentiation of NETs by TEM.

2. Materials and methods

2.1. Neutrophil harvesting

Mouse neutrophils were kindly supplied by Dr. J.

Thalhammer and Dr. R. Weiss (Department of Molecular

Biology, University of Salzburg, Austria). Peripheral blood

mononuclear cells (PBMCs) were prepared from whole blood

by erythrocyte lysis under gently hypotonic conditions. Two

millilitre of heparin-treated whole mouse blood were mixed

W.D. Krautgartner, L. Vitkov / Micron 39 (2008) 367–372368

with 5 ml of FACS Lysing Solution (Becton Dickinson,

Schwechat, Austria) and incubated at room temperature for

10 min. Seven millilitre of the nutritive medium (MEM, PAA,

Pasching, Austria) were added and the cells were centrifuged

for 10 min at 1200 rpm. The supernatant was discarded and the

pellet was washed two times in 10 ml MEM. Finally, PBMCs

were resuspended in 1 ml MEM, supplemented with 1% fetal

calf serum, 1% L-glutamine, 1% penicillin/streptomycin, 1 mM

sodium pyruvate, 20 mM HEPES, 1% nonessential aminoacids

and 2 mM beta-mercaptoethanol. The final concentration of the

freshly separated mouse PBMCs was 2 � 107 cells/ml, whereof

10–60% were neutrophils. Twenty microlitre of the cell

suspension were dropped on each grid (100 mesh gold grids

covered by formvar). Subsequently, the grids were placed into a

humid chamber at 37 8C for 90 min. The grids were blotted and

dropped with 20 ml of the same nutritive solution supplemented

by 25 nM PMA (phorbol 12-myristate 13-acetate, LC

Laboratories, Woburn, MA 01801) at 37 8C for 60 min to

induce the NET formation (Brinkmann et al., 2004).

2.2. DNA digestion

Half of the samples were treated with 20 mM Tris–HCl and

5 mM MgCl2 and the rest with 1 mg/ml deoxyribonuclease

(DNase I, Roche Diagnostics GmbH, Mannheim, Germany)

buffered with 20 mM Tris–HCl and supplemented with 5 mM

MgCl2 at 378 for 60 min.

2.3. Osmium tetroxide (Os) staining

The grids were washed with 0.1 M sodium cacodylate

buffered at pH 5.6. Fixation was performed by 1.2%

glutaraldehyde buffered at pH 5.6 with 0.1 M sodium cacodylate

for 15 min at room temperature. Subsequently, the grids were

treated with 1.2% osmium tetroxide buffered at pH 5.6 with

0.1 M sodium cacodylate for 1 min at room temperature.

2.4. Osmium tetroxide–uranyl acetate–lead citrate

(Os–U–Pb) staining

Half of the grids stained with osmium tetroxide were

subsequently stained with 1% aqueous uranyl acetate (Ultros-

tain 1, Leica, Vienna, Austria) as well as 1% aqueous lead

citrate (Ultrostain 2, Leica, Vienna, Austria) by LKB 2168

Ultrostainer (LKB Produkter AB, Bromma, Sweden).

2.5. Cuprolinic blue (CB) staining

The grids were washed with 0.05 M sodium acetate buffered

at pH 5.6. Subsequently, the grids were fixed with 1.2%

glutaraldehyde and 0.2% cuprolinic blue (Cuprolinic blue,

Polysciences Inc., Warrington, PA 18976) (buffered at pH 5.6

with 0.05 M sodium acetate) in dark for 30 min at room

temperature. Enhancement was performed with 1% aqueous

sodium tungstate for 30 s and subsequent dehydration was

performed with an ascending series of ethanol. The 30 and 50%

ethanol drops contained also 1% sodium tungstate.

2.6. Ruthenium red-osmium tetroxide (RR-OsO4) staining

The grids were washed with 0.1 M sodium cacodylate

buffered at pH 5.6. Subsequently, the grids were fixed with

1.2% glutaraldehyde and 0.05% ruthenium red (buffered at pH

5.6 with 0.1 M sodium cacodylate) for 15 min at room

temperature. Postfixation was performed with 1.2% osmium

tetroxide (buffered at pH 5.6 with 0.1 M sodium cacodylate)

and 0.05% ruthenium red for one min at room temperature.

After staining, all of the grids were simply washed and air-

dried. The RR-OsO4 drop-on-grid specimens and particularly

the drop-on-grid cuprolinic blue specimens were unstable and

faded after a few days.

2.7. TEM processing

All samples were examined with a transmission electron

microscope LEO EM 910 (LEO Elektronenmikroskopie Ltd.,

Oberkochen, Germany) operating at 80 kV.

3. Results

Os-stained samples revealed no structures. Os–U–Pb

staining revealed well formed, but faintly blackened networks

consisting of a multitude of interwoven threads. They branched

into thinner threads, but no fibrils with a diameter 15–17 nm

could be distinguished (Fig. 1). CB staining revealed a similar

appearance of NETs, however, somewhat better staining was

achieved (Figs. 2–4). RR-OsO4 staining showed at low

magnification a similar appearance of NETs (Fig. 5). However,

the higher magnification revealed that the thinner threads

branched into fine fibres (Fig. 6). A sharp demarcation of

deeply blackened fibres with diameters of circa 15–17 nm and

less frequently of circa 6 nm was evident (Fig. 7), however, no

globular domains could be observed.

Two predominant patterns of thread alignment were

observed: a radiating one and a meshwork-like one. The

radiating thread alignment pattern was characterized by the

branching of a few thick threads into thinner ones, which on

their part branched into fibres (Figs. 5–7). The meshwork-like

pattern covered areas extending to more than 1000 mm2 (Fig. 8)

and was characterized by a relative uniformity of appearance of

dense interwoven threads and fibres with a diameter of circa

15–17 nm (Fig. 9). In contrast, fibres with a diameter of 6 nm

were not observed within this pattern.

The treatment by DNase prior to the fixation completely

disaggregated the NETs. In the RR-OsO4 stained samples,

however, a multitude of tiny particles, very probably NET

fragments, were observed (Fig. 10). The electron density of the

particles is the same as that of the NETs (Fig. 11).

4. Discussion

Morphologically, NETs have been characterized as DNase-

labile net-like formations of fibres with a diameter of 15–

17 nm, with so-called globular domains on them with a

diameter of approximately 25 nm (Brinkmann et al., 2004).

Figs. 1–4. (1) Os–U–Pb staining. NETs. Inset: The higher magnification shows lack of fine structural details. (2) CB staining. An overview of NETs. Some similarity

with light microscopy micrographs (Brinkmann et al., 2004) is evident. (3) CB staining (a detail of Fig. 2). The deeply blackened spots (arrows) are probably

phthalocyanine precipitates, as previously reported (Krautgartner et al., 2003, 2005). (4) CB staining (a detail of Fig. 3). The higher magnification shows lack of fine

structural details.

W.D. Krautgartner, L. Vitkov / Micron 39 (2008) 367–372 369

Besides the bactericidal effects caused by neutrophil elastase

and histones, the ability to trap bacteria is another key

characteristic of NETs. The bacterial binding by NETs

probably occurs by fimbriae-mediated adhesion. Indeed,

bacterial fimbriae-mediated adhesion to histones (Zhu et al.,

2005) and to DNA (van Schaik et al., 2005) has been

demonstrated. Both DNA and bacterial fimbriae are poorly

depicted by routine TEM staining techniques. As DNA and

microbial fimbriae are polyanions, cationic stains have been

used for their visualization by TEM. Thus, uranyl and lead ions

(Pearse, 1985; Trendelenburg and Puvion-Dutilleul, 1987), in

particular the combination of uranyl acetate and lead citrate, are

still constantly employed for this purpose. However, these

staining techniques have low specificity, feebly contrast the

DNA and therefore disable a good resolution. Two cationic

dyes, the phthalocyanine cuprolinic blue and ruthenium red

have also been used for chromatin staining.

CB has been used for visualizing DNA in light microscopy

(Tas et al., 1983; Mendelson et al., 1983), however, enhancing

the phthalocyanine staining by sodium tungstate enables the

attainment of a sufficient contrast in TEM (Scott et al., 1981).

Despite the good staining specificity, the DNA contrast attained

by this technique was insufficient to achieve high resolution.

Nevertheless, the possibility to visualize the microbial adhesion

via phthalocyanines at TEM level (Fassel et al., 1992;

Krautgartner et al., 2003, 2005) makes this method, albeit

restricted by its limited resolution, suitable to study the NETs-

bacterial interactions.

Ruthenium red shows a strong affinity for chromatin

(Stockert and Pelling, 1992; Engelhardt, 2000). However, as

chromatin and also NETs consist of two main components,

DNA and proteins, it remains unclear which component(s)

really stain by RR-OsO4. Thus, it has been hypothesized by

Engelhardt (Engelhardt, 2000) that the chromatin staining by

RR-OsO4 could be caused by a reaction with the chromatin

glycoproteins (Reeves et al., 1981; Turner et al., 1990).

Although RR alone produces some degree of electron opacity

(Stockert and Pelling, 1992), RR-OsO4 creates a much more

intense contrasting. Indeed, the presently employed RR-OsO4

staining technique enabled the outstanding staining of the

Figs. 5–7. (5) RR-OsO4 staining. An overview of NETs. Some similarity with light microscopic micrographs (Brinkmann et al., 2004) is evident. (6) RR-OsO4

staining (a detail of Fig. 5). The alignment of the threads is clearly distinguishable. (7) RR-OsO4 staining (a detail of Fig. 6). Fibres with diameter of 15–17 nm form a

meshwork. Arrows: some fibres with a diameter of 6 nm are also evident.

W.D. Krautgartner, L. Vitkov / Micron 39 (2008) 367–372370

NETs, despite the superimposition by cellular debris and

proteins from the nutritive solution. The RR-positive NETs

mainly consisted of fibres with a diameter of circa 15–17 nm,

which were interwoven in net-like formations. RR-OsO4

staining revealed also fibres with a diameter of circa 6 nm,

which has not been observed with scanning electron micro-

scopy. RR-OsO4 stains microbial fimbriae (Fassel et al., 1992;

Fassel and Edmiston, 1999; Vitkov et al., 2001, 2002, 2005a,b),

Figs. 8–11. (8) RR-OsO4 staining. Huge areas are covered by meshwork of a relatively uniform density. (9) RR-OsO4 staining. The higher magnification reveals no

basic differences to the loose networks. Inset: Interwoven fibres with similar dimension as shown in Fig. 7. (10) RR-OsO4 staining. The sample is treated by

deoxyribonuclease prior to the staining. No networks, but multitudes of tiny particles were observed. (11) RR-OsO4 staining (a detail from Fig. 10). Many deeply

contrasted particles, probably NET fragments, are evident. The blackening of the particles is the same as that of the NETs.

W.D. Krautgartner, L. Vitkov / Micron 39 (2008) 367–372 371

proteoglycans (Chan and Wong, 1992; Rodgers et al., 1995) and

chromatin. Consequently, the DNase digestion of the NET

fibres verified their DNA character. By contrast, no globular

domains were visualized via RR-OsO4; consequently, they lack

chromatin. Two modes of NET spreading were observed:

radiating ones and web-like ones. In the radiating NETs, the

threads were branched into fibres with a diameter of 15–17 nm.

The threads have been suggested to consist of aggregated fibres

(Brinkmann et al., 2004). In the web-like NETs, the spread was

more homogenous and fewer threads with smaller diameters

were observed. In our opinion, the web-like appearance was a

consequence of the superimposition of a multitude of better

spread NETs. As the adhesion of NETs to the formvar causes a

transformation of the three-dimensional NETs into two-

dimensional images, deductions concerning the three-dimen-

sional relationship between the NET elements by virtue of the

presented results are restricted. The ability of RR staining to

visualize the microbial adhesion makes this method particu-

larly appropriate for studying the NETs-bacterial interactions.

The deeply blackened DNase-sensitive smooth stretches, with

their characteristic diameter, are the assertive criterion to

identify NETs.

5. Conclusions

The employed RR-OsO4 staining technique enables the

visualization of both NETs and fimbriae-mediated bacterial

adhesion (Vitkov et al., 2001, 2002, 2005a,b). It thus facilitates

the examination of NETs-bacterial interactions at TEM level.

The combination of the following two characteristics guarantees

the high specificity of this staining procedure: the blackening by

RR-OsO4 and the disintegration of the RR-OsO4-positive net-

like structures by DNase. Additionally, the combination of the

specific fibre thickness (15–17 nm) and of the deep blackening of

the fibres is a very strong indication of NETs.

Acknowledgements

The authors thank Professor Josef Thalhammer and Dr.

Robert Weiss for supplying the mouse neutrophils, Dr. Karin

W.D. Krautgartner, L. Vitkov / Micron 39 (2008) 367–372372

Oberascher, Mrs. Adda Maenhardt and Mrs. Michaela

Klappacher for the technical assistance as well as Mr. Andreas

Zankl for the image processing.

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