Real-Time, High-Throughput Microscopic Quantification of Human Neutrophil Extracellular Trap Release and Assessing the Pharmacology of Antagonists

We developed a real-time microscopy method for the quantification of neutrophil extracellular traps or NETs. These NETs play an important role in innate immune defense. However, it has become clear that accumulation of NETs in tissues contributes to the pathophysiology of multiple inflammatory and autoimmune diseases.

Because of the pathological implications of NETs, interest in the development of NETosis antagonists has risen. To study NETosis inhibition, we developed a real-time microscopy method for the quantification of human NET release, allowing us to study NETosis and its inhibition in a high-throughput manner. Neutrophils are sensitive cells and can change their responsiveness to stimuli during the purification process due to mechanical, microbial and other types of stress.

Therefore, it is important to validate the neutrophil isolation protocol to minimize neutrophil activation. So this technique enables the study of NET release from healthy as well as diseased individuals. In addition, it allows us to study the kinetics of NET formation induced by different physiological stimuli.

With this high-throughput microscopy technique, we were able to demonstrate that the NETosis antagonist CIT-013, a monoclonal antibody targeting citrullinated histones 2A and 4, was able to efficiently inhibit NET release with an IC 50 or 4.6 nanomolar. This demonstrates that this assay is suitable for testing NETosis Antagonists. After collecting peripheral blood from the healthy volunteer in a lithium heparin tube, transfer the blood to a fresh 50 milliliter tube.

Rinse the lithium heparin tube with DPBS and transfer it to the same 50 milliliter tube, ensuring the blood to DPBS ratio is 1:1. After mixing, add diluted blood on top of the density gradient solution. Centrifuge the blood at 400 G for 40 minutes with minimal acceleration and break.

Discard the top plasma layer with a 10 milliliter pipette. Then, use a plastic pasteur pipette to discard the peripheral blood mononuclear cells and the density gradient solution. Gently shake the layer containing erythrocyte and neutrophil before re-suspending it in 15 milliliters of DPBS.

Add 25 milliliters of 6%dextran and 0.9%sodium chloride solution and mix by inverting the tube. After 25 minutes of incubation, transfer the top neutrophil layer to a fresh 50 milliliter tube with a 10 milliliter pipette and centrifuge at 500 G for 10 minutes. Decant the tube to discard the supernatant and re-suspend the pellet in 10 milliliters of ammonium chloride potassium lysis buffer.

Add 40 milliliters of the same lysis buffer and incubate at room temperature while continuously inverting the tube until the solution becomes translucent. Centrifuge the tube at 350 G for 10 minutes. After removing the supernatant, slowly and dropwise, add five milliliters of culture medium, 10%on top of the neutrophil pellet without bringing the neutrophils into suspension.

Gently swirl the tube until most of the erythrocytes present on top of the neutrophil pellet are re-suspended in the culture medium. After removing the supernatant, re-suspend the neutrophil pellet in 10 milliliters of culture medium, 10%Once fully re-suspended, add up to 50 milliliters of culture medium 10%and centrifuge the mixture before re-suspending the pellet in 10 milliliters of Neutrophil Extracellular Trap or NET assay buffer. To begin, add 0.001%Poly-L-lysine solution to each well of the 96-well plate and incubate for one hour at 37 degrees Celsius.

Wash the wells three times with 200 microliters of DPBS and air dry the wells. Next, re-suspend the required number of neutrophils in the Neutrophil Extracellular Trap or NET assay buffer. Transfer four times concentrated DNA dye in NET assay buffer to each well.

Add four times concentrated NETosis stimuli in NET assay buffer to the corresponding wells. Then, add four times concentrated NETosis antagonists in NET assay buffer and neutrophil suspension to the corresponding wells. Centrifuge the plate at 100 G for two minutes.

Then, insert the plate in the live cell microscopy analysis system placed in an incubator at 37 degrees Celsius and 5%carbon dioxide. After placing the 96-well imaging plate containing neutrophil suspension in a live cell microscopy analysis system, open the system software. Click schedule to acquire and click the plus button to launch.

Then, select scan on schedule and click next. To create a new vessel from scratch, select new in the create vessel section and click next. In the Scan Type section, select Standard, and click Next.

Select the scan settings as cell by cell, none. Image channels, phase contrast in green. Acquisition time, 100 milliseconds and objective 20x.

Then click Next. From the Vessel Selection section, select the appropriate type of vessel to scan and click Next. Specify the location in the drawer for the vessel and click Next.

In the Scan Pattern section, select the wells that need to be scanned and the number of images per well. Click Next. In the Vessel Notebook section, provide information about the vessel.

Enter the plate name and click Next. In the Analysis Setup section, select defer analysis until later and click Next. In the Scan Schedule section, select create new schedule with scans at intervals of, and select one hour in the Add Scans to Schedule section.

Select Stop scanning at five hours after the first scan. Click Next and click Add to schedule when the scanning information is correct. After acquiring phase contrast and immunofluorescence images of neutrophils, launch the live cell microscopy analysis software.

From the view, select the live cell microscopy experiment to be analyzed. Select Create New Analysis Definition and click Next. Then, select basic analyzer from the Analysis Type section and click Next.

In the Image Channel section, select green, deselect phase, and click Next. Open the image layers window, select green and deselect phase. Turn off autoscale in the green section and manually set min and max.

Then open the vessel scan times window and select the images representing positive control wells with NETs. Open the analysis definition settings window and write NETs for the object name. For the segmentation, select Adaptive.

For the threshold GCU, select between three and five, and set the Edge Split On between minus 10 and zero. Next, for cleanup, select Hole Fill to 100 square micrometers and adjust size to minus one pixel. From Filters, select area greater than 200 square micrometers, Mean Intensity less than 24.6, and Integrated Intensity greater than 7, 000.

Then click preview current or preview all to apply the settings. Hover over the different NET structures and adjust the analysis settings to fine tune the false positive and false negative NETs. When the settings for the NET analysis are correct, click Next.

Select the time points and wells to analyze in the scan times and well section and click Next. Insert the definition name in the Save and Apply Analysis Definition section and click Next. Click Finish when the analysis information is verified and correct.

Open the experiment analysis by clicking the analysis within the vessel of interest. Then, open the graph metrics window. Next, click the plus button to create a metric.

When presenting data as percentage NET confluency, select Area in the Metric section and select Confluence in the Value section. When presenting data as a percentage of NETting neutrophils, select Object Count in the Metric section and select Per image in the Value section. After configuring Metric settings, in the user-defined Metrics section, select NETs Area Confluence or NETs Object Count Per Image.

Select all required time points and wells for analysis from the Select Scans and Select Well sections respectively. Finally, click Export Data. Calcium ionophore stimulation induced faster NETosis compared to PMA, which resulted in a higher proportion of neutrophils releasing NETs.

NETs induced by calcium ionophores were more diffuse beyond the neutrophil plasma membrane, while PMA-induced NETs remained closer to the neutrophil plasma membrane. A trend towards elevated NET levels was observed when neutrophils were activated with coated immune complexes, FMLP and monosodium urate crystals. NET release in response to a 23187 was completely inhibited by CIT-013 at a half maximal inhibitory concentration of 4.6 nanomolar.