Induction and Visualization of Neutrophil Extracellular Traps in Pre-Labeled Neutrophils

Begin with a multi-well plate containing fluorescently labeled neutrophils and treat them with patient serum containing anti-neutrophil cytoplasmic antibodies.

These antibodies interact with the neutrophil's Fc receptors, activating a signaling pathway, producing reactive oxygen species or ROS, and activating peptidyl arginine deiminase.

ROS causes degranulation of neutrophils, releasing enzymes — elastase and myeloperoxidase into the cytoplasm. These enzymes and activated peptidyl arginine deiminases translocate to the nucleus.

Elastase cleaves histone-H1, and myeloperoxidase modifies histones, initiating chromatin decondensation. Activated peptidyl arginine deiminase induces histone citrullination, converting arginine residues to citrulline and contributing to further decondensation.

The decondensed chromatin mixes with intracellular proteins, including antimicrobial and cytoplasmic proteins. Later, the neutrophil's membrane disintegrates, releasing decondensed chromatin mixed with cytoplasmic proteins into the extracellular space, forming a neutrophil extracellular trap, NET.

Add a cell-impermeable DNA dye, binding exclusively to extracellular DNA in NETs. Under a fluorescence microscope, neutrophils appear red with green extracellular web-like structures, confirming NET formation.