Simplified flow cytometric quantification of human neutrophil extracellular traps (NETs)

Area of Science:

• Immunology
• Cell Biology
• Biochemistry

Background:

• Neutrophil extracellular traps (NETs) are formed through NETosis, a cell death pathway distinct from apoptosis and necrosis.
• Existing methods for quantifying NETosis are limited in efficiency and can be prone to errors and subjectivity.

Purpose of the Study:

• To develop a simple, sensitive, reliable, and flexible flow cytometry assay for efficient detection and quantification of NETosis.
• To validate the assay's accuracy and its ability to distinguish NETosis from other neutrophil activation and cell death processes.

Main Methods:

• Isolated primary human neutrophils were stimulated and fixed.
• Fixed cells were incubated with anti-DNA/Histone 1 and anti-histone H2A antibodies for dual fluorescent detection.
• Imaging flow cytometry was used for validation.

Main Results:

• The assay successfully detected and quantified NETosis by identifying double-fluorescent cells (H1-DNA/H2A).
• The method accurately discriminated NETosis from neutrophil activation, apoptosis, and necrosis.
• Established NETosis pathways were confirmed using known inducers.

Conclusions:

• The novel flow cytometry assay provides a robust and adaptable tool for NETosis research.
• This methodology reduces human error and subjectivity compared to existing techniques.
• The assay facilitates further investigation into NETosis pathways and associated disease pathogenesis.