An In Vitro Technique to Generate Human Neutrophil Extracellular Traps

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. NETs Generation

1. Stimulate neutrophils with 500 nM of PMA (per 30 ml of neutrophil solution) and incubate on a 150 x 25mm flat tissue culture dish with a 20 mm grid for 4 hr at 37 °C 5% CO₂. This will allow NETosis.
2. After 4 hr of stimulation, gently aspirate and discard the media, leaving the layer of NETs and neutrophils adhered at the bottom. Do not disrupt this layer.
3. Using a total of 15 ml of cold PBS without Ca and Mg per dish, wash the bottom of each dish by pipetting 15 ml of PBS on the bottom of the dish in order to lift off all adherent material from the bottom.
4. Collect solution obtained from washing each dish (step 1.3) in a 15 ml conical tube and centrifuge for 10 min at 450 x g at 4 °C. Neutrophils and any remaining cells will pellet at the bottom, leaving a cell-free NET-rich supernatant.
5. Divide supernatant into 1.5 ml micro-centrifuge tubes and spin for 10 min at 18,000 x g at 4 °C. This will allow all DNA to pellet.
   NOTE: Centrifugation in larger tubes is easier and less time-consuming if such high speeds are attainable on the regular centrifuge, otherwise will need to divide supernatants in smaller tubes in order to use high-speed micro-centrifuge.
6. Discard the supernatant and resuspend all pellets obtained together in ice-cold PBS to a concentration corresponding to 2 x 10⁷ neutrophils per 100 μl of PBS. This will yield the cell-free NET stock that can be used for subsequent experiments.
7. Measure DNA concentration in the sample obtained using spectrophotometry or an alternate DNA quantification tool. An adequate concentration in the sample should range between 140 – 180 ng/μl.