Quantification of Neutrophil Extracellular Traps Isolated From Mouse Tissues

Abstract

One of the most intriguing functions of neutrophils is the production of neutrophil extracellular traps (NETs), which are formed when neutrophils decondense their internal DNA and extrude it along with cytotoxic proteins in a web‐like structure. This process allows neutrophils to trap and kill pathogens, and is also associated with multiple hematological and autoimmune conditions. Due to their rapid degradation, there are many challenges in accurately and specifically detecting and quantifying NETs. Microscopy is the gold standard for NET detection, but is not optimal for large‐scale screening. Furthermore, methods relying on detection of free DNA or on flow cytometry–based examination of NET‐associated markers can be nonspecific, time‐consuming, and expensive. Here, we describe an innovative, quick, specific, and inexpensive conventional flow cytometry method for detecting neutrophils on the verge of forming NETs. These methods utilize pulse‐shaped analysis (PulSA) to distinguish resting neutrophils from those with decondensed DNA, a prerequisite for NET formation. An increase in DNA‐diffuse neutrophils is found in cell populations after exposure to NET‐inducing stimuli, consistent with the DNA decondensation expected during neutrophil NET formation. These populations are only observed in granulocytes, validating the specificity of this method. We describe protocols optimized for neutrophils retrieved from mouse blood, spleen, and bone marrow. The relative speed and simplicity of the method described here makes it a useful tool for detecting NET formation in large‐scale experiments. © 2020 Wiley Periodicals LLC. Basic Protocol: Detection of nuclear decondensation in neutrophils from stimulated murine bone marrow Alternate Protocol 1: Detection of nuclear decondensation in neutrophils from splenocytes Alternate Protocol 2: Detection of nuclear decondensation in neutrophils from blood Support Protocol 1: Cryopreservation and defrosting of samples Support Protocol 2: Paraformaldehyde fixation of samples

The Role of Neutrophil Extracellular Traps in the Outcome of Malignant Epitheliomas: Significance of CA215 Involvement

“…This approach provides better insight into the sequential aspects, architecture, or heterogeneity of NET production. However, it is more challenging to analyze and typically requires a well-trained researcher to fully integrate and corelate the data within the experimental or clinical context [81,90,[96][97][98][99]. These assays require a multi-faceted approach.…”

“…A combination of methods provides a comprehensive qualitative analysis of NETs, revealing their formation, structure, components, and interactions in various contexts [81,90,[96][97][98][99].…”

Emerging Role of Neutrophil Extracellular Traps in Gastrointestinal Tumors: A Narrative Review

“…In addition, Rebecca et al reported a method that can preliminarily judge whether NETs are generated. The method utilizes pulse-shaped analysis (PulSA) to distinguish resting neutrophils from those with decondensed DNA, which is a prerequisite for NET formation [185]. Therefore, we can rule out the generation of NETs in patients and their associated pathological analysis by detecting the absence of neutrophils with decondensed DNA.…”