This article discusses the formation of neutrophil extracellular traps (NETs), which are structures that trap and neutralize pathogens. The study outlines the methods for inducing NET formation in vitro using phorbol myristate acetate (PMA) and details the cellular mechanisms involved.
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.
To stimulate the formation of neutrophil extracellular traps, or NETs, add 500 nanomolar of PMA to 30 milliliters of the neutrophil suspension in a 150-millimeter by 25-millimeter flat tissue culture dish with a 20-millimeter grid. Incubate the cells for four hours at 37 degrees Celsius under 5% carbon dioxide.
After incubation, gently aspirate and discard the medium, taking care not to disrupt the layer of NETs and neutrophils adhered to the bottom of the dish. Then, use a pipette to wash the bottom of each dish with 15 milliliters of cold PBS without calcium and magnesium. All adherent material should be removed from the bottom.
Collect the wash suspension in a 15-milliliter conical tube and then centrifuge at 450 x g for 10 minutes at 4 degrees Celsius. Neutrophils and any remaining cells will pellet at the bottom, leaving a cell-free, NET-rich supernatant.
Next, decant the supernatant into multiple 1.5-milliliter tubes and centrifuge at 18,000 x g for 10 minutes at 4 degrees Celsius to pellet the DNA. Discard the supernatant, and re-suspend the pellet in PBS such that the concentration corresponds to 2 x 107 neutrophils per 100 microliters of PBS. This cell-free NET stock can be used for subsequent experiments.
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