A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

We developed a high-throughput assay to measure neutrophil extracellular traps, or NETs. NETs are immunogenic DNA structures that are exposed by neutrophils. NETs can trap and kill microorganisms and are therefore an important anti-infectious mechanism, but they also play a pathogenic role in autoimmune diseases.

With this technique, NETs are quantified by three-dimensional immunofluorescence confocal microscopy resulting in a highly sensitive and high-throughput method to study NET formation and degradation. This technique enables us to quantify NET formation of patients with autoimmune diseases. Ex vivo NET formation of patients with ANCA-associated vasculitis or systemic lupus erythematosis could be monitored with this assay.

In addition, potential therapeutics targeting NET formation could be tested with this assay in vitro. Neutrophil isolation could be a struggle for people who never have performed this technique before. To begin this procedure, obtain 20 milliliters of peripheral blood from a healthy donor in two 10-milliliter EDTA-coded tubes.

Transfer 10 milliliters of blood in a sterile 50-milliliter tube and PBS to a final total volume 32.5 milliliters. Using a 10-milliliter pipette and pipette controller, take up 14 milliliters of density gradient. Place the pipette on the bottom of the 50-milliliter tube.

Then, take the pipette controller off the pipette, allowing the density gradient to flow out by gravity until the maximum is reached by capillary effect. Place a thumb on top of the pipette to prevent the remaining density gradient from leaking out, and then remove the pipette from the tube. Centrifuge the tube at 912 times g at room temperature for 20 minutes without acceleration or brake.

Carefully remove the white ring containing the peripheral blood mononuclear cells first, followed the PBS-diluted plasma. Lastly, remove the density gradient layer as much as possible. Next, retrieve a bottle of cold sterile distilled water and a flask of 10x concentrated PBS from a refrigerator.

Working quickly, add 36 milliliters of the water directly on top of the pellet and carefully mix once. After 20 seconds counted from the start, add four milliliters of 10x PBS to make an isotonic solution. And again, mix once carefully.

Centrifuge the tube at 739 times g and at four degrees Celsius for five minutes. Discard the supernatant, making sure to be extremely careful as the pellet is not solid, and quickly repeat the process of adding the cold sterile distilled water and concentrated PBS as previously described. Centrifuge at 328 times g and four degrees Celsius for five minutes.

Carefully remove the supernatant and re-suspend the pellet in five milliliters of PBS. Count the neutrophils and keep them on ice. First, make a neutrophil suspension containing approximately 10 to 20 million neutrophils in two milliliters of PBS in a 15-milliliter tube.

In a different 15-milliliter tube, mix two milliliters PBS with four microliters of two-micromolar red fluorescent cell linker. Gently add the red fluorescent cell linker solution to the neutrophil suspension and mix carefully. Wrap the tube in aluminum foil to protect the mixture from light, and incubate for exactly 25 minutes at 37 degrees Celsius to label the neutrophils with the red fluorescent cell linker.

Inactivate the labeling by adding RPMI 1640 medium containing 10%heat inactivated FCS at room temperature to a final volume of 15 milliliters and mix once carefully. If after this a pellet has formed, carefully re-suspend the mixture. Centrifuge the tube at 328 times g at room temperature for five minutes.

Before removing the supernatant, ensure that a pellet has formed. After this, re-suspend the pellet in five milliliters of red-free RPMI 1640 medium containing 2%FCS at room temperature. Then, count the neutrophils.

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 two, four, or six hours. Then, prepare the volume of impermeable DNA dye needed to add 25 microliters of five-micromolar impermeable DNA dye to reach a final concentration of one micromolar in each well.

15 minutes before the end of the incubation time, add 25 microliters of five-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 three 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 one 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 Image J 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 afterwards. Presented here is a highly-sensitive broadly applicable assay for the semi-automated quantification of neutrophil extracellular trap formation for the evaluation of ex vivo induction of neutrophil extracellular traps upon different stimuli. NET formation is quantified in a three-dimensional manner by quantifying stained extracellular DNA over 10 Z-stacks with three-micrometer distance starting off at the focal plane in each well.

By measuring the cumulative area, the sensitivity of the assay increases. The total area of stained neutrophils imaged are quantified only in the focal plane in each well which correlates significantly with the total neutrophil count in each well with a Pearson correlation coefficient of 0.99. The representative outcome of quantification of NET formation in neutrophils is expressed as cumulative stained extracellular DNA area over 10 Z-stacks per imaged neutrophil.

Snapshots are then taken of the net quantification assay. Representative images of unstimulated neutrophils and of NETs in AAV-stimulated neutrophils are shown here with the fluorescent-labeled neutrophils in red and the stained extracellular DNA in green. Neutrophils should be handled with care, and the PKH labeling should be performed exactly according to the protocol.

This assay can be used to study morphology, kinetics, and composition of NETs. This technique is another method that provides the possibility to study NETs in health and disease. Trypan blue, PKH, and PFA should be handled with gloves and should not be breathed in directly.