In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

This method can answer key questions about neutrophil extracellular trap or NET formation in dogs or other species. The main advantages of these techniques includes the real live detection of NET formation and identification of NET components like mild peroxidase and citrullinated histone H3.Begin by obtaining 10 to 20 milliliters of blood from the jugular vein of a healthy canine donor, removing the needle from the syringe before dispensing the blood into sodium heparin tubes to avoid excessive sharing. Gently invert the tubes a few times to ensure an adequate mixing of the blood and anticoagulant, and check for clots and red cell aggregates before placing the samples on ice.

Dilute the anticoagulated blood with an equal volume of ice cold Dulbecco’s PBS, and transfer eight milliliters of diluted blood into a 50 milliliter polypropylene conical tube containing 25 milliliters of filtered 3%Dextran. Place the tube upright for 30 minutes at room temperature to allow aggregation and sedimentation of the erythrocytes. Then carefully layer five milliliters of leukocyte-rich plasma on top of five milliliters of density gradient medium in a 14 milliliter polypropylene round-bottom tube.

After density gradient centrifugation, use a five milliliter serological pipette to transfer the intermediate granulocyte cell layer into 50 milliliter conical, and lyse any erythrocytes co-aspirated with the polymorphonuclear cells with 20 milliliters of cold, ultrapure water. Gently invert the tube for 30 to 60 seconds to ensure adequate lysing before washing the cells in an equal volume of ice cold 1.8%sodium chloride solution. After inverting the tube a few times, centrifuge at four degrees Celsius.

Use a serological pipette to carefully remove the supernatant, and gently resuspend the pellet in 100 microliters of cell culture buffer. Count and confirm their viability by trypan blue exclusion. Then dilute the neutrophils to a one times ten to the six cells per milliliter of cell culture buffer concentration.

It is essential to use a proper sterile technique and to handle the neutrophils carefully throughout the isolation process as contamination and shear forces can lead to in vitro necrosis and cell lysis. To image the neutrophils by fluorescence microscopy, add 200 to 400 microliters of cells into each well of a poly-L-lysine coated 12-well culture plate for a 30 minute incubation at 37 degrees Celsius. When the neutrophils have attached to the bottom of the wells, label the cells with a suitable fluorescent nucleic acid dye for 10 minutes at room temperature.

At the end of the incubation, activate the neutrophils with 100 micrograms per milliliter of LPS, 100 nanomolar of PMA as a positive control, or an equivalent volume of cell culture buffer as the negative control at 37 degrees Celsius. Then image the neutrophil cultures by fluorescence microscopy at a 40 times magnification at the indicated time points. For a NET quantification, carefully place 18 millimeter diameter poly-D-lysine coated coverslips into individual wells of a 12-well culture plate and label the wells according to the experimental stimulus.

Then seed 100 to 200 microliters of the isolated neutrophils directly onto the coverslips for a 30-minute incubation at 37 degrees Celsius. When the neutrophils have adhered to the coverslips, activate the neutrophils in each well with LPS, PMA, or DPBS for 180 minutes as just demonstrated. During the last 30 minutes of the incubation, layer a paraffin film sheet over a test tube stand to create shallow wells, and add two to three drops of 4%paraformaldehyde to each well.

Label the wells as experimentally appropriate, and use fine forceps to gently transfer the coverslips from each plate well upside down onto the appropriate corresponding PFA filled well. After 20 minutes at room temperature, wash the fixed cells three times for five minutes in two to three drops of buffered saline per wash by moving the cells from one well of DPBS to the next. To permeabilize the cells, gently lay the coverslips upside down on a drop of 1%NP-40 for one minute followed by three one-minute buffered saline washes on a rocker.

After the last wash, place the coverslips upside down in 100 to 200 microliters of 5%goat serum in individually labeled petri dishes lined with fresh plastic paraffin film for a one-hour incubation at 37 degrees Celsius with rocking. At the end of the blocking incubation, wash the coverslips three times in buffered saline with rocking as just demonstrated. Transfer the coverslips into individual plastic paraffin film-lined petri dishes in 50 to 100 microliters of the appropriate antibody of interest for a one-hour incubation at 37 degrees Celsius with rocking.

After washing, incubate the coverslips with the appropriate fluorophore-conjugated secondary antibody for one hour at 37 degrees Celsius protected from light. At the end of the secondary antibody incubation, wash the coverslips three times in buffered saline in the dark with rocking followed by nucleic acid dye-staining with DAPI for five minutes in the dark at room temperature. After three one-minute buffered saline washes in the dark with rocking, apply a 50 microliter drop of antifade mounting medium onto one glass slide per sample, and carefully mount each dried coverslip upside down onto one drop of mounting medium.

Then allow the samples to cure overnight at four degrees Celsius protected from light. The next morning, obtain 10 images randomly from different regions of each coverslip at a 40 times magnification. All intact neutrophils, regardless of treatment, should appear green and exhibit the characteristic lobulated nuclei at zero to 30 minutes after stimulation.

At around 90 minutes, the nuclei of PMA-treated neutrophils begin to lose their lobulated appearance and continue to decondense until the release of cell-free DNA. LPS, a slower and more physiologic stimulant of canine neutrophils, typically does not result in chromatin decondensation or NETosis until 90 minutes after stimulation. By 120 minutes, LPS and PMA-activated neutrophils can be seen surrounded by cell-free DNA.

Compared to LPS-stimulated cells more PMA-activated neutrophils lose their plasma membrane integrity as evidenced by their staining with cell impermeant dye. Unstimulated neutrophils should maintain their lobulated nuclei and intact plasma membranes throughout the incubation period. Neutrophils stimulated by LPS for 180 minutes, produce discreet web-like NET scaffolds in close proximity to nearby neutrophils.

In contrast, PMA-activated neutrophils produce vast amounts of NETs that are noticeably larger in area. Indeed, PMA, a potent stimulant of an enzyme that catalyzes the essential NET component intracellular citrullinated histone H3, induces a higher number of intracellular citrullinated histone H3 expressing cells compared to unstimulated and LPS-stimulated neutrophils. While performing this assay, it is extremely important that the operator acquiring and analyzing the microscope images to be blinded to the experimental conditions to maintain objective evaluation of the assay.

Half of these procedures have their assays, like the ELISA or flow cytometry, can be performed to further quantify citrullinated histone H3 in cells. Don’t forget to include negative controls consisting of either unstimulated neutrophils or neutrophils treated with the peptidyl arginine inhibitor, chrolide amidine. We also recommend testing different dilutions of primary and secondary antibodies to minimize nonspecific binding and interferences.