A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

The overall goal of this experiment is to isolate quiescent neutrophils and measure their DNA release in response to agonists. This method can help answer key questions in immunology and veterinary medicine such as, what is the neutrophil response to various agonists of neutrophil extracelluar trap induction or cell death? The main advantage of this techinique is that it allow the rapid screening of various agonists and inhibitors of neutrophil extracellular trap formation.

Begin by inserting a 21-gauge butterfly needle into the cephalic vein of a healthy canine donor then attach a three-milliliter syringe and obtain three three-milliliter blood samples, immediately transferring the samples into uncapped EDTA-containing blood tubes. Take care that the venipuncture is atraumatic and that the blood flow is good, as a poor blood draw will result in a poor neutrophil yield. After filling each tube, invert the tubes to ensure an adequate mixing of the blood and anti-coagulant.

Pool the blood in a 50-milliliter conical tube. Mix the samples with an equal volume of sterile room-temperature 3%Dextran 500 by gentle inversion and leave the tube upright for 18 to 20 minutes at room temperature. Then transfer the straw-colored upper layer to a fresh tube until it can no longer be collected without contamination by the lower red layer.

Centrifuge the upper layer. Discard the supernatant, manually resuspending the pellet in 10 milliliters of sterile room-temperature PBS. Next, add 10 milliliters of a room-temperature polysucrose in sodium diatrizoate cell separation solution to a 50-milliliter conical tube.

Carefully layer the cells over the separation solution. Separate the cells by centrifugation. Discard the upper three plasma and platelet mononuclear cell and density gradient layers.

Lyse any of the remaining erythrocytes with 10 milliliters of room-temperature water. After 30 seconds, add 10 milliliters of sterile room-temperature 1.8%sodium chloride to restore the tonicity. Mix the cells by gentle inversion.

Now collect the cells by centrifugation. Gently resuspend the white pellet in 10 milliliters of sterile PBS. After counting, centrifuge the cells again and resuspend the pellet at a five times 10 to the sixth cells per milliliter concentration in sterile room-temperature PBS.

As appropriate, air-dry a small volume of cells on a glass slide, followed by staining with a rapid fixation and staining kit according to the manufacturer's instructions. If the neutrophil isolation has been successful, at least 95%of the nucleated cells should be granulocytes with minimal erythrocyte contamination as observed under light microscopy. Immediately after the neutrophil isolation, pass the cell suspension through a sterile 70-micron sterile filter and add five times 10 to the fourth cells to the test wells of a 96-well plate containing culture media for a 30 minute incubation at 39 degrees celsius and 4%carbon dioxide.

When the cells have attached, add up to a final volume of 100-200 microliters of the agonist of interest in RPMI without phenol red and supplemented with heat-inactivated FCS to the test wells, as well as to two blank wells containing PBS alone. Then add an appropriate cell-impermeable nucleic acid dye to the cells and return the plate to the incubator. After the appropriate incubation period, measure the fluorescence on a microplate reader.

A one-hour stimulation of canine neutrophils with platelet-activating factor results in a mean four-fold increase in fluorescence compared with control non-stimulated cells. PMA however, is a slower agonist that does not demonstrate an increased neutrophil fluorescence before two hours of activation, but which ultimately produces a similar fold increase over time. Nucleic acid dye fluorescence is not specific for neutrophil extracellular trap formation, as it will also occur if there is cell death by other mechanisms or if the reagents are contaminated with DNA.

For example, in this representative experiment, a commercial LPS preparation produced very high fluorescence in the absence of neutrophils, presumably due to contamination with bacterial DNA. Once mastered, this technique can be completed in approximately six hours if it is performed properly. While attempting this procedure, it's important to remember to handle the neutrophils gently.

Do not vortex the cells, and pipette off rather than pour off the supernatants. Following this procedure, other methods, like immunofluorescence microscopy, can be performed to confirm that the DNA released correlates to NETosis. After its development, this technique paved the way for researchers in the field of hematology to explore the interaction of DNA released on coagulation and the effect of novel NET agonists including patient plasma.

After watching this video, you should have a good understanding of how to isolate canine neutrophils and measure their response to agonists.