An In Vitro Technique to Generate Human Neutrophil Extracellular Traps

Neutrophil extracellular traps, or NETs, are web-like structures of chromatin fibers complexed with antimicrobial proteins that trap and neutralize pathogens. NETs are secreted by neutrophils — immune cells that store antimicrobial proteins in specialized cytoplasmic granules.

To study NET formation in vitro, take a suspension of isolated neutrophils. Add phorbol myristate acetate, or PMA — a lipophilic pro-inflammatory compound. Plate the cells, and incubate.

PMA crosses the neutrophil membrane and enters the cytoplasm, where it binds to and activates protein kinase C or PKC.

Activated PKC induces downstream signaling to phosphorylate cytoplasmic components of NADPH oxidase — an enzyme complex — causing its assembly on the plasma and granule membrane. The complex produces reactive oxygen species or ROS.

The ROS cause the release of proteases and antimicrobial proteins from the granules. Granular proteases, along with ROS-activated citrullination enzymes, translocate to the nucleus.

The granular proteases cause partial histone degradation, while the citrullination enzymes promote histone citrullination — neutralizing positive charges. These modifications disrupt the histone-DNA interactions, resulting in chromatin decondensation.

The modification of the nuclear lamina causes nuclear envelope rupture and chromatin externalization; the chromatin mixes with the cytoplasmic antimicrobial proteins, forming NETs. The permeabilization of the cell membrane causes the extracellular release of NETs.

Discard the medium without disturbing the NETs and neutrophils adhered at the bottom. Resuspend the adhered components in a buffer.

Centrifuge to precipitate the cells — leaving a NET-rich supernatant ready for downstream assays.