Induction and Visualization of Neutrophil Extracellular Traps in Pre-Labeled Neutrophils

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Isolation of Healthy Neutrophils

1. Obtain 20 mL of peripheral blood from a healthy donor in two 10 mL ethylenediaminetetraacetic acid (EDTA)-coated tubes.
2. Put 10 mL of blood in a sterile 50 mL tube and add phosphate-buffered saline (PBS) up to 32.5 mL.
3. Add density gradient (e.g., Ficoll-amidotrizoaat) under the cells.
   1. Take up 14 mL of density gradient with a 10 mL pipet and pipet controller.
   2. Place the pipet on the bottom of the 50 mL tube.
   3. Take the pipet controller off the pipet, allowing the density gradient to flow out of the pipet by gravity until the maximum is reached by capillary effect (1-2 mL will be left), without using the motor.
   4. Remove the pipet by placing a thumb on top of the pipet, thereby preventing the density gradient from leaking out during the removal of the pipet.
4. Spin the tubes for 20 min at 912 x g and room temperature (RT) without acceleration or brake.     
   NOTE: Red blood cells (RBC) and neutrophils have a high density and are at the bottom of the 50 mL tube. Peripheral blood mononuclear cells (PBMCs) are separated and on top of the density gradient as a white ring. PBS-diluted plasma will be above the PBMCs. If needed, PBMCs can be isolated by transferring the white ring to a new 50 mL tube with additional washing steps with phosphate-buffered saline (PBS).
5. Carefully remove the white ring containing PBMCs first, followed by the removal of the PBS-diluted plasma, and lastly the density gradient layer as much as possible.
6. To isolate neutrophils from the neutrophil/erythrocytes mix, lyse erythrocytes with cold sterile distilled water.
   1. Take a cold sterile distilled water bottle and a 10x concentrated PBS flask from the fridge.
   2. Work quickly for this step. Add 36 mL of cold sterile distilled water directly on top of the pellet and mix once carefully. Add 4 mL of 10x PBS after 20 s to make an isotonic solution. Mix once carefully.
   3. Spin tube for 5 min at 739 x g and 4 °C (for the removal of RBCs). Neutrophils will be in the white pellet.
   4. Carefully discard the supernatant. Perform step 1.6.2 again and make sure the pellet is suspended properly.
   5. Spin tube for 5 min at 328 x g and 4 °C.
   6. Carefully remove the supernatant and resuspend the pellet in 5 mL of PBS. Count the neutrophils and keep them on ice.    
      NOTE: Expected yield from 1 tube of blood (10 mL) is approximately 15-75 million neutrophils.

2. Red Fluorescent Cell Labelling of Neutrophils

1. Make a neutrophil suspension of 10-20 million neutrophils in 2 mL of PBS in a 15 mL tube.
2. Make a solution of 2 mL PBS with 4 µL of 2 µM red fluorescent cell linker (see Table of Materials) in a different 15 mL tube. Add this gently to the neutrophil suspension and mix carefully.
3. Incubate in the dark for exactly 25 min at 37 °C to label the neutrophils with the red fluorescent cell linker.
4. Inactivate the labeling by adding Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) at RT up to 15 mL and mix once carefully. Make sure that the pellet is carefully resuspended if a pellet has formed.
5. Spin tube for 5 min at 328 x g and RT.
6. Remove the supernatant and resuspend the pellet in 5 mL of phenol red-free RPMI 1640 medium containing 2% FBS and 10% Penicillin/streptomycin (P/S) at RT. Count the neutrophils.
   NOTE: A cell loss of 50% can occur after red fluorescent cell labeling.

3. Induction of Neutrophil Extracellular Trap Formation

1. Make a cell suspension of 0.42 x 10⁶ cells/mL in phenol red-free RPMI 1640 medium containing 2% FBS and 10% P/S.
2. Add 37,500 neutrophils in 90 µL per well in a black 96-well, flat bottom plate.
3. Add 10 µL of the chosen stimulus (e.g., sera of a patient) in triplicate to reach a concentration of 10% in the well. Always include a negative control (medium) in triplicate.
4. Incubate in the dark at 37 °C for the desired time, ranging from 30 min to 2, 4, or 6 h. Incubating for 4 hours is suggested.
5. Calculate the volume needed to add 25 µL of 5 µM impermeable DNA dye (see Table of Materials), to reach a final concentration of 1 µM in the well. Make a predilution if necessary, in RPMI 1640 medium containing 2% FBS and 10% P/S to obtain a 5 µM concentration.
6. Add 25 µL of 5 µM impermeable DNA dye 15 min before the end of the incubation time. Continue incubation for another 15 min at 37 °C in the dark.
7. Remove the supernatant (~125 µL) very carefully and store it if needed. Add 100 µL of 4% paraformaldehyde (PFA). Keep in the dark, and immediately continue with step 4.

4. Neutrophil extracellular traps (NET) Visualization with 3D High-content, High-resolution Immunofluorescence Confocal Microscopy

1. Configure the settings on the immunofluorescence confocal microscope by clicking on the Acquisition setup.
   1. Click on the Configure tab.
   2. Select the objective and the camera. Choose the 10X Apo Lambda objective with an acquisition mode of a confocal 60 µm pinhole.
   3. Click on Plate and choose the 96-well plastic plate. Select the sites to visit and choose a fixed number of sites. Fill in 3 columns and 3 rows without overlap (0 µm), which cover a total of 45% of the well.
   4. Click on Acquisition. Select Enable Laser-Based Focusing. Select Acquire Z Series/Time Series if needed.
   5. Click on Site Autofocus.
      1. Click on Focus on Plate Bottom | Off-Set by Bottom Thickness. For the initial well to find the sample, choose the first well acquired. For the site autofocus, choose all sites.
   6. Click on Wavelengths.
      1. For the number of wavelengths, choose 2. For the TL shading correction refinement level, choose 2.
      2. For Wavelength 1, select Texas Red.
         1. For the autofocus options, select laser with Z offset, post laser offset 1.1 µm. Use Z-stack with a custom range of 200-10.
      3. For Wavelength 2, select fluorescein isothiocyanate (FITC).
         1. For the autofocus options, select laser with Z offset from w1 0 µm. Use Z-stack with a custom range of 200-10.
         2. For the acquisition options, select Z series and 2D projection image maximum. For the acquisition options, select Shading Correction | Off.
   7. Select Z Series. Select the number of steps: 10. Select the step size: 3 μm (total range will be 27 µm).
2. Put the plate in the immunofluorescence confocal microscopy.
   1. Click on the Run tab.
      1. Fill out the plate name and description and choose the storage location.
      2. Select the wells that need to be acquired.
3. Choose exposure time for Texas Red and FITC.
4. Click on Acquire Plate to start the acquisition, which will take approximately 1 h per plate.

5. Analysis of NET Formation

1. Use an image processing program designed for the analysis of scientific multidimensional images (see Table of Materials) to analyze NET formation.
2. Transfer the acquired image data to a separate hard drive.
3. Select the color-adding tool.
   1. Select w1 and choose the folder in the hard drive where the data is stored.
   2. Select w2 and choose the folder in the hard drive where the data is stored.
      NOTE: Use a standard macro that uses w1 in the file name to add red color to the Texas Red images and uses w2 to add green color to the FITC images.
4. Select Analysis Macro.
   1. Select w1, and choose the threshold value (intensity threshold), which is usually 10. Select the desired pixel value (size threshold, e.g., 100).
   2. Select w2, and choose the threshold value (intensity threshold), which is usually 10. Select the desired pixel value (size threshold, e.g., 500).
   3. Choose the destination for the spreadsheet file, run the analysis, and save log files afterward.
5. Analyze data in a spreadsheet.