Real-Time High Throughput Technique to Quantify Neutrophil Extracellular Traps Formation in Human Neutrophils

Our research focuses on neutrophils, which are essential first responders in the immune system and play a significant role in various diseases. Over the past two decades, it has become clear that neutrophils contribute to the development of cancer or immune diseases and other inflammatory conditions by disrupting immune regulation. This includes forming neutrophil extracellular traps, NETs, web-like structures that respond to inflammation, and could be targeted for therapy in these diseases.

However, despite some promising molecules targeting NETs in development, there is still no approved therapy that specifically affects this mechanism. This is at least partially attributable to the lack of an objective, unbiased, reproducible, and high-throughput quantification method for NET formation. Our protocol employs dual color live cell imaging to analyze neutrophil behavior using membrane-permeable and impermeable dyes for precise NET formation tracking.

This method distinguishes between NET forming and healthy neutrophils based on membrane integrity and provides clear differentiation of cell death types through morphological changes observed in phase contrast imaging. This method overcomes the problems of previously reported techniques to quantify NET formation and provides an efficient, reproducible, and accurate NET quantification in an automated manner. This method will help in neutrophil-targeted drug development by giving the ability to screen multiple therapeutic targets against NET formation in a high throughput manner.

After isolating neutrophils from human peripheral blood, resuspend them in RPMI 1640 medium in a 1.5 milliliter centrifuge tube. Add one microliter of membrane-permeable red DNA dye to 1.5 milliliters of neutrophil suspension, and incubate in the dark for five minutes. Centrifuge the neutrophil suspension at 2, 500G for five minutes at room temperature.

After centrifugation, remove the tube containing pelleted neutrophilsn and aspirate the supernatant. Resuspend the neutrophils in one milliliter of RPMI and centrifuge it again. After the last wash, resuspend the neutrophils in one milliliter of RPMI.

Add a small volume of neutrophil suspension to the hemocytometer, and count them under a microscope. Dilute the neutrophil suspension to 1.5 times 10 to the fifth neutrophils per milliliter using RPMI. Add four microliters of one to 100 pre-diluted membrane-impermeable green DNA dye to the one milliliter of neutrophil suspension.

Dispense 100 microliters of neutrophil suspension per well into a clear tissue culture treated 96 well plate. Add 100 microliters of RPMI-containing stimulus reagents with or without inhibitor into the respective wells. Place the plate in a live cell analysis system housed in a 5%carbon dioxide incubator.

To begin, start the live cell analysis system software. Press the plus symbol at the top left corner of the screen. to initiate the add vessel.

Choose Scan on Schedule, then select New to run the scanning program. Select Standard. Then, set scan settings as cell by cell options to none, and image channels to phase, green, and red with respective acquisition times.

Set objective to 20X. From the list, choose the plate and select the vessel location to put the plate on the tray of the imaging system. Select the wells containing samples and decide the number of images to capture per well.

This information automatically generates an estimated scan duration for the plate. Click Create Plate Map to input information for each well, such as cell type and compound. Then, name the study.

On the next screen, select basic analyzer as the analysis type, and choose analysis definition from the dropdown list, which shows previously used analysis definitions. Leave spectral unmixing for both green and red at 0.0. Schedule the scan to occur every 15 to 20 minutes for eight hours.

Drag the white and gray bar on top of the screen to set the starting time of the scan. On the next screen, review the scan settings, and press Add to Schedule to start scanning. After scanning, on the view tab, open the vessel for analysis, press Launch Analysis on the left of the screen and select Create New Analysis Definition.

Choose basic analyzer and use phase, green, red, and overlap image channels. Select six to eight representative sample images for training. To configure the analysis definition, set green objects to neutrophils forming neutrophil extracellular traps or NETs, and red objects to the nuclei of all neutrophils.

Press Preview Current or Preview All and adjust each parameter to optimize the results. Choose the scan times and wells for analysis. Then, provide a label for the analysis definition.

Review the summary and launch the analysis. After analysis, double click on the name of the analysis definition to open the analyzed study. Open layers on the left of the screen and check if each cell is correctly marked.

Click Graph Metrics on the left of the screen, then select count per image for green count. Choose the time points and wells to be exported. For select grouping, select Plate Map Replicates to confirm all wells are correctly specified during scanning, and click on Export Data.

Similarly, export the data for red count and overlap count. Using the given equation, calculate the percentage of NET-forming cells for each condition and time point. The time course of the NET formation is visualized by plotting the percentage of NET-forming neutrophils at each time point.

Adding an AKT inhibitor blocked the NET formation in neutrophils stimulated with PMA, or calcium ionophore, demonstrating that potential molecules targeting NET formation may be tested in a high-throughput manner using this methodology.