This article describes a method for studying neutrophil extracellular trap (NET) formation using fluorescently labeled neutrophils and patient serum containing anti-neutrophil cytoplasmic antibodies. The process involves activating neutrophils, leading to chromatin decondensation and NET release, which can be visualized under a fluorescence microscope.
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.
Make a cell suspension at a density of 420,000 cells per milliliter in phenol red-free RPMI-1640 medium containing 2% FCS. Add 37,500 neutrophils in 90 microliters to each well of a black 96-well flat-bottom plate.
Next, add 10 microliters of the chosen stimulus, in triplicate, to reach a concentration of 10% in each well. Always include a negative control in triplicate. Incubate in the dark at 37 degrees Celsius for the desired time, ranging from 30 minutes to 2, 4, or 6 hours. Then, prepare the volume of impermeable DNA dye needed to add 25 microliters of 5-micromolar impermeable DNA dye to reach a final concentration of 1 micromolar in each well.
15 minutes before the end of the incubation time, add 25 microliters of 5-micromolar impermeable DNA dye to each well. Continue the incubation for the final 15 minutes at 37 degrees Celsius in the dark. After this, remove the supernatant very carefully and store if needed. Add 100 microliters of 4% paraformaldehyde. Keep the plate in the dark, and immediately proceed to the next section.
After configuring the settings, select Z Series. Select 10 as the Number of Steps. Select 3 micrometers as the Step Size. Load the plate into the immunofluorescence confocal microscope.
Click on the Run tab. Fill out the plate name and description and choose the storage location. Select the wells that need to be acquired. Choose the Exposure Time for Texas Red and FitC, then click on Acquire Plate and start the acquisition, which will take approximately 1 hour per plate.
After this, select Analysis Macro. Then, select W1 and choose the threshold value. Next, select the desired pixel value, and finally, in a second ImageJ window, select W2 and choose the threshold value with the desired pixel value. Select a destination for the spreadsheet file. Run the analysis, and save the log file afterward.
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