In Vitro Stimulation and Visualization of Macrophage Extracellular Traps

Begin with an adherent macrophage culture.

Add pro-inflammatory cytokines.

The cytokine binds to the macrophage surface receptors, initiating a series of inflammatory events.

First, the cytokine signaling triggers the production of antimicrobial peptides.

Simultaneously, the chromatin within the macrophage nucleus undergoes decondensation.

This process, coupled with changes in membrane composition, disrupts the nuclear membrane, releasing decondensed chromatin into the cytoplasm.

In the cytoplasm, the antimicrobial peptide interacts with the released chromatin.

Cytokine signaling-mediated cell membrane permeabilization causes the decondensed chromatin to be released into the extracellular space, forming a Macrophage Extracellular Trap, or MET.

These web-like structures consist of DNA, histones, antimicrobial molecules, and enzymes.

Add SYTOX green, a fluorescent nucleic acid dye that selectively binds to the exposed DNA in the extracellular traps.

Under a fluorescence microscope, the MET appears as green streaks of extracellular DNA released from the stimulated macrophage.

Under sterile conditions, prepare the culture media containing different stimulators of MET release to the complete RPMI 1640 media. For experiments with hypochlorous acid stimulation, prepare 200 micromolar hypochlorous acid in a Falcon tube with HBSS that's been pre-warmed to 37 degrees Celsius.

After the polarization treatment, aspirate the cell media from each well, and carefully wash the cells three times with 1-milliliter aliquots of either sterile PBS for PMA, TNF-alpha, and interleukin-8 stimulation, or HBSS for hypochlorous acid stimulation.

After removing the PBS in the final washing step, add 1 milliliter of complete media containing PMA, TNF-alpha, or interleukin-8. Incubate the cells at 37 degrees Celsius and 5% carbon dioxide in a cell incubator for six hours for TNF-alpha stimulation, and 24 hours for PMA or interleukin-8 stimulation.

For experiments with hypochlorous acid, after removing the HBSS in the final washing step, add 1 milliliter of freshly prepared hypochlorous acid. Then, incubate the cells for 15 minutes at 37 degrees Celsius in the presence of 5% carbon dioxide in a cell incubator. After that, carefully aspirate the cell supernatant, and wash the cells three times with 1-milliliter aliquots of HBSS.

After removing the HBSS from the final wash step, add 1 milliliter of complete RPMI 1640 culture media. Then, incubate the cells for 24 hours at 37 degrees Celsius in the presence of 5% carbon dioxide in a cell incubator.

Prepare SYTOX green dye in HBSS at a concentration of 40 micromolar. Directly add 25 microliters of the dye to each well containing HMDM. Incubate cells at room temperature for 5 minutes in the dark. Then, place the HMDM in tissue culture wells on the microscope stage of an inverted fluorescent microscope for imaging.

Turn on a broad spectrum fluorescent light source, bright field light source, and inverted microscope installed with a high-resolution color digital camera. Rotate the filter wheel to the number 2 position for green fluorescence with excitation at 504 nanometers and emission at 523 nanometers.

Using the 5X objective, focus the image with the coarse focus, then the fine focus knobs on the microscope until the image appears sharp, clear, and focused. Switch the microscope to camera mode.

Start the associated software. Click the Play button to preview the image, and adjust the fine focus knob on the microscope until the image appears sharp, clear, and focused in the software preview window. Click the Capture button.

Within the software, click File, Save as the required image file type. On the microscope, rotate the filter wheel to the number 5 position for brightfield imaging, and repeat the capture procedures to obtain the corresponding brightfield image.