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Neutrophil Extracellular Traps: How to Generate and Visualize Them
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Visualizing NETosis Using a Novel Neutrophil Extracellular Trap-Specific Marker.

Meghan L De Meo1,2,3, Muhammad H Shahzad1,2, Jonathan D Spicer4,5,6,7

  • 1Thoracic Surgery & Upper Gastrointestinal Cancer Research Laboratories, McGill University, Montreal, QC, Canada.

Methods in Molecular Biology (Clifton, N.J.)
|December 31, 2022
PubMed
Summary

A novel marker for histone H3 cleavage improves visualization of neutrophil extracellular traps (NETs). This method identifies more NETs at earlier stages, offering superior detection compared to current techniques.

Keywords:
3D9Confocal fluorescent microscopyHistone H3 citrullinationHistone H3 cleavageHistone H3 clippingImmunofluorescenceMyeloperoxidaseNETosisNeutrophilNeutrophil extracellular trap

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Area of Science:

  • Immunology
  • Cell Biology
  • Pathophysiology

Background:

  • Neutrophils are key leukocytes involved in homeostasis and disease.
  • Neutrophil extracellular traps (NETs) contribute to inflammatory, non-infectious, and neoplastic pathologies.
  • Current NET visualization methods lack specificity and sensitivity.

Purpose of the Study:

  • To introduce a novel, NET-specific marker for improved NETosis visualization.
  • To compare the efficacy of the new marker against conventional NET detection methods.
  • To elucidate a stepwise mechanism of NETosis involving histone H3 cleavage.

Main Methods:

  • Identification of a novel NET-specific posttranslational modification: N-terminal histone H3 cleavage.
  • Comparative analysis of the novel marker versus co-staining (myeloperoxidase and citrullinated histone H3) for NET visualization.
  • Investigation of the temporal relationship between histone H3 cleavage and citrullination during NETosis.

Main Results:

  • A single marker of histone H3 N-terminal cleavage is superior to conventional co-stains for visualizing NETs.
  • The novel marker identifies significantly more NETs and detects their formation at earlier stages.
  • A stepwise NETosis mechanism is proposed, with histone H3 cleavage preceding citrullination.

Conclusions:

  • A novel histone H3 cleavage marker provides enhanced visualization of NETosis.
  • This method offers improved sensitivity and specificity for NET detection.
  • The findings facilitate further research into the role of NETs in immunity and disease.