Oral Biology pp 481-497 | Cite as

Characterization, Quantification, and Visualization of Neutrophil Extracellular Traps

  • Phillipa C. White
  • Ilaria J. Chicca
  • Martin R. Ling
  • Helen J. Wright
  • Paul R. Cooper
  • Mike R. Milward
  • Iain L. C. ChappleEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1537)


Following the discovery of neutrophil extracellular traps (NETs) in 2004 by Brinkmann and colleagues, there has been extensive research into the role of NETs in a number of inflammatory diseases, including periodontitis. This chapter describes the current methods for the isolation of peripheral blood neutrophils for subsequent NET experiments, including approaches to quantify and visualize NET production, the ability of NETs to entrap and kill bacteria, and the removal of NETs by nuclease-containing plasma.

Key words

Neutrophil extracellular traps Reactive oxygen species Fluorescence microscopy SEM Chemiluminescence DNA Elastase Myeloperoxidase Cathepsin G Immunostaining 


  1. 1.
    Borregaard N, Cowland JB (1997) Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 89:3503–3521PubMedGoogle Scholar
  2. 2.
    Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, Zychlinsky A (2004) Neutrophil extracellular traps kill bacteria. Science 303:1532–1535CrossRefPubMedGoogle Scholar
  3. 3.
    Bianchi M, Niemiec MJ, Siler U, Urban CF, Reichenbach J (2011) Restoration of anti-Aspergillus defense by neutrophil extracellular traps in human chronic granulomatous disease after gene therapy is calprotectin-dependent. J Allergy Clin Immunol 127:1243–1252CrossRefPubMedGoogle Scholar
  4. 4.
    Wang Y, Li M, Stadler S, Correll S, Li P, Wang D, Hayama R, Leonelli L, Han H, Grigoryev SA, Allis CD, Coonrod SA (2009) Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol 184:205–213CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Yousefi S, Mihalache C, Kozlowski E, Schmid I, Simon HU (2009) Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps. Cell Death Differ 16:1438–1444CrossRefPubMedGoogle Scholar
  6. 6.
    Pilsczek FH, Salina D, Poon KK, Fahey C, Yipp BG, Sibley CD, Robbins SM, Green FH, Surette MG, Sugai M, Bowden MG, Hussain M, Zhang K, Kubes P (2010) A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus. J Immunol 185:7413–7425CrossRefPubMedGoogle Scholar
  7. 7.
    Harris PC (2012) Effect of density gradient material upon ex-vivo neutrophil behaviour, and effect of neutrophil extracellular traps upon the growth and survival of periodontopathogenic bacteria. MRes thesis, University of BirminghamGoogle Scholar
  8. 8.
    Fuchs TA, Abed U, Goosmann C, Hurwitz R, Schulze I, Wahn V, Weinrauch Y, Brinkmann V, Zychlinsky A (2007) Novel cell death program leads to neutrophil extracellular traps. J Cell Biol 176:231–241CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Palmer L, Cooper PR, Ling MR, Wright HJ, Huissoon A, Chapple IL (2012) Hypochlorous acid regulates neutrophil extracellular trap release in humans. Clin Exp Immunol 167:261–268CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Beiter K, Wartha F, Albiger B, Normark S, Zychlinsky A, Henriques-Normark B (2006) An endonuclease allows Streptococcus pneumoniae to escape from neutrophil extracellular traps. Curr Biol 16:401–407CrossRefPubMedGoogle Scholar
  11. 11.
    Delbosc S, Alsac JM, Journe C, Louedec L, Castier Y, Bonnaure-Mallet M, Ruimy R, Rossignol P, Bouchard P, Michel JB, Meilhac O (2011) Porphyromonas gingivalis participates in pathogenesis of human abdominal aortic aneurysm by neutrophil activation. Proof of concept in rats. PLoS One 6, e18679CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Behrendt JH, Ruiz A, Zahner H, Taubert A, Hermosilla C (2010) Neutrophil extracellular trap formation as innate immune reactions against the apicomplexan parasite Eimeria bovis. Vet Immunol Immunopathol 133:1–8CrossRefPubMedGoogle Scholar
  13. 13.
    Byrd AS, O’Brien XM, Johnson CM, Lavigne LM, Reichner JS (2013) An extracellular matrix–based mechanism of rapid neutrophil extracellular trap formation in response to Candida albicans. J Immunol 190:4136–4148CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Saitoh T, Komano J, Saitoh Y, Misawa T, Takahama M, Kozaki T, Uehata T, Iwasaki H, Omori H, Yamaoka S, Yamamoto N, Akira S (2012) Neutrophil extracellular traps mediate a host defense response to human immunodeficiency virus-1. Cell Host Microbe 12:109–116CrossRefPubMedGoogle Scholar
  15. 15.
    Keshari RS, Jyoti A, Dubey M, Kothari N, Kohli M, Bogra J, Barthwal MK, Dikshit M (2012) Cytokines induced neutrophil extracellular traps formation: implication for the inflammatory disease condition. PLoS One 7, e48111CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Palmer LJ (2010) Neutrophil extracellular traps in periodontitis. Ph.D. thesis, University of BirminghamGoogle Scholar
  17. 17.
    Berends ET, Horswill AR, Haste NM, Monestier M, Nizet V, von Köckritz-Blickwede M (2010) Nuclease expression by Staphylococcus aureus facilitates escape from neutrophil extracellular traps. J Innate Immun 2:576–586CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Wartha F, Beiter K, Albiger B, Fernebro J, Zychlinsky A, Normark S, Henriques-Normark B (2007) Capsule and D‐alanylated lipoteichoic acids protect Streptococcus pneumoniae against neutrophil extracellular traps. Cell Microbiol 9:1162–1171CrossRefPubMedGoogle Scholar
  19. 19.
    Lauth X, von Köckritz-Blickwede M, McNamara CW, Myskowski S, Zinkernagel AS, Beall B, Ghosh P, Gallo RL, Nizet V (2009) M1 protein allows Group A streptococcal survival in phagocyte extracellular traps through cathelicidin inhibition. J Innate Immun 1:202–214CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Urban CF, Reichard U, Brinkmann V, Zychlinsky A (2006) Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms. Cell Microbiol 8:668–676CrossRefPubMedGoogle Scholar
  21. 21.
    Hakkim A, Fürnrohr BG, Amann K, Laube B, Abed UA, Brinkmann V, Herrmann M, Voll RE, Zychlinsky A (2010) Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis. Proc Natl Acad Sci U S A 107:9813–9818CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Leffler J, Martin M, Gullstrand B, Tydén H, Lood C, Truedsson L, Bengtsson AA, Blom AM (2012) Neutrophil extracellular traps that are not degraded in systemic lupus erythematosus activate complement exacerbating the disease. J Immunol 188:3522–3531CrossRefPubMedGoogle Scholar
  23. 23.
    Zhou L, Somasundaram R, Nederhof RF, Dijkstra G, Faber KN, Peppelenbosch MP, Fuhler GM (2012) Impact of human granulocyte and monocyte isolation procedures on functional studies. Clin Vaccine Immunol 19:1065–1074CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Das T, Sehar S, Manefield M (2013) The roles of extracellular DNA in the structural integrity of extracellular polymeric substance and bacterial biofilm development. Environ Microbiol Rep 5:778–786CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Phillipa C. White
    • 1
  • Ilaria J. Chicca
    • 1
  • Martin R. Ling
    • 1
  • Helen J. Wright
    • 1
  • Paul R. Cooper
    • 1
  • Mike R. Milward
    • 1
  • Iain L. C. Chapple
    • 1
    Email author
  1. 1.Institute of Clinical Sciences, College of Medical and Dental Sciences, The School of DentistryUniversity of BirminghamBirminghamUK

Personalised recommendations