An In Vitro Technique to Generate Human Neutrophil Extracellular Traps

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.