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Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT
Published on: November 27, 2019
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An Imaging and Computational Algorithm for Efficient Identification and Quantification of Neutrophil Extracellular
Apurwa Singhal1, Shubhi Yadav1, Tulika Chandra2
1Pharmacology Division, CSIR-Central Drug Research Institute, Lucknow 226021, India.
Cells
|January 21, 2022
Summary
Neutrophil extracellular traps (NETs) are implicated in numerous diseases. A new high-content screening method offers rapid, specific, and automated detection of NETs, overcoming limitations of previous techniques.
Area of Science:
- Immunology
- Cell Biology
- Computational Biology
Background:
- Neutrophil extracellular traps (NETs) are increasingly recognized in various pathologies like sepsis, autoimmune diseases, and COVID-19.
- Current methods for NET detection are often subjective, non-specific, error-prone, and lack high throughput, hindering therapeutic development.
- Inconsistent identification, nomenclature, and quantification of NETs impede targeted therapeutic strategies.
Purpose of the Study:
- To develop and validate an innovative, high-throughput imaging and computational algorithm for the precise detection and analysis of NETs.
- To overcome the limitations of existing methods, enabling rapid, specific, and unbiased quantification of NET formation.
- To establish a platform for identifying NET formation inhibitors and modulators of neutrophil death pathways.
Main Methods:
- Utilized a high-content screening (HCS)-cellomics platform with membrane-permeable and impermeable DNA dyes for in situ NET detection.
- Developed an automated algorithm for single-cell analysis, assessing nuclear morphology, area, and intensity changes.
- Combined with Annexin V staining to differentiate NETs from apoptosis and necrosis, without fixation or permeabilization steps.
Main Results:
- The HCS-based algorithm enabled rapid, specific, and automated detection of NET-forming cells.
- Automated analysis minimized user bias and accurately distinguished NETs from other cell death modalities.
- The method allows for time-dependent monitoring of NET formation due to the absence of fixation/permeabilization.
Conclusions:
- The developed imaging and computational approach provides a robust, high-throughput platform for NET analysis.
- This method facilitates the discovery of novel therapeutic agents targeting NET formation or modulating neutrophil death.
- This technology may offer new strategies for resolving inflammation by targeting NETosis-apoptosis pathways.
