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Related Experiment Video

Updated: Nov 21, 2025

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation
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Quantification of Neutrophil Extracellular Traps Isolated From Mouse Tissues.

Rebecca L Buckland1, Alicia S Wilson1, Anne Brüstle1

  • 1John Curtin School of Medical Research, The Australian National University, Canberra, Australia.

Current Protocols in Mouse Biology
|January 15, 2021
PubMed
Summary

Researchers developed a novel flow cytometry method to detect neutrophils preparing to form neutrophil extracellular traps (NETs). This quick and specific technique aids in studying NET formation in various conditions.

Keywords:
PulSAflow cytometryneutrophilneutrophil extracellular traps

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Area of Science:

  • Immunology
  • Cell Biology
  • Hematology

Background:

  • Neutrophil extracellular traps (NETs) are crucial for pathogen defense but also implicated in autoimmune diseases.
  • Accurate detection of NETs is challenging due to their rapid degradation and limitations of current methods like microscopy and nonspecific flow cytometry markers.
  • Existing methods are often time-consuming, expensive, and not suitable for large-scale screening.

Purpose of the Study:

  • To develop a rapid, specific, and cost-effective flow cytometry method for detecting neutrophils undergoing NETosis.
  • To utilize pulse-shaped analysis (PulSA) to differentiate neutrophils with decondensed DNA (pre-NETosis) from resting neutrophils.
  • To provide optimized protocols for analyzing neutrophils from mouse blood, spleen, and bone marrow.

Main Methods:

  • Conventional flow cytometry utilizing pulse-shaped analysis (PulSA).
  • Distinguishing neutrophils based on nuclear DNA decondensation, a key step in NET formation.
  • Optimized protocols for mouse neutrophils from blood, spleen, and bone marrow, including fixation and cryopreservation support.

Main Results:

  • An increase in DNA-diffuse neutrophils was observed in cell populations upon exposure to NET-inducing stimuli.
  • The observed DNA decondensation is consistent with the expected process during neutrophil NET formation.
  • The method demonstrated specificity, with DNA-diffuse populations exclusively found in granulocytes.

Conclusions:

  • The developed PulSA-based flow cytometry method offers a quick, specific, and inexpensive approach to detect neutrophils on the verge of NET formation.
  • This technique is suitable for large-scale experiments and aids in studying NETosis in various biological contexts.
  • The method provides a valuable tool for research in immunology, hematology, and autoimmune disease.