In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

The release of extracellular traps by neutrophils and other immune cells is important in innate immunity but also strongly linked with inflammatory pathologies. Very little is known about this process in macrophages although these cells play a critical role in inflammatory response. This protocol provides a primary human in vitro model of macrophages extracellular trap or mast release.

It provides a model for us to study a potential physiological growth of this structure in vivo. The main advantage of this technique is that the cells from this protocol are primary cells isolated from human buffy coat preparations. There is no priming step required to differentiate the monocyte into macrophages, which is contrast with other monocyte cell line such as THP-1 cell line.

This protocol is easily adapted to other immune cells including isolated neutrophil and microglia. Extracellular trap produced by macrophages are very fragile, so cares must be taken between step to preserve the integrity of the stained traps. Under sterile conditions, prepare M1 priming medium by adding to the complete RPMI-1640 culture media, interferon gamma to the concentration of 20 nanograms per milliliter and lipopolysaccharide to the concentration of one microgram per milliliter.

Prepare M2 priming media by adding interleukin 4 to the complete RPMI-1640 culture media to the concentration of 20 nanograms per milliliter. Under sterile conditions, aspirate media from the seeded and cultured tissue culture plate wells containing HMDM. Carefully add into each well, one milliliter of sterile PBS pre-warmed to 37 degrees Celsius.

Remove the PBS buffer and wash two additional times. Add one milliliter of either the M1 or M2 priming media to each well containing the HMDM. Incubate the cells for 48 hours at 37 degrees Celsius in the presence of 5%carbon dioxide in a cell incubator.

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 one 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 one 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 one 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 cells’supernatant and wash the cells three times with one milliliter aliquots of HBSS. After removing the HBSS from the final wash step, add one 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 two position for green fluorescence with excitation at 504 nanometers and dimension 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 five position for bright-field imaging, and repeat the capture procedures to obtain the corresponding bright-field image.

Bright-field images showing the morphological changes of HMDM in response to stimuli are shown here. M1-polarized macrophages from experiments with HMDM exposed to interferon gamma and lipopolysaccharide showed an elongated and spindle-like cell shape. For comparison, the morphology of the M2-polarized macrophages after exposure of HMDM to interleukin 4 for 48 hours were typically round and flat.

The ability of differentiated HMDM phenotypes to release METs was visualized by live-cell fluorescence imaging with SYTOX green. The control obtained from each HMDM phenotype incubated for 24 hours in the absence of any pro-inflammatory stimuli showed very limited green staining. Positive staining for METs, shown as green streaks, was achieved with exposure of M1-HMDMs to hypochlorous acid, PMA, interleukin 8, or TNF alpha.

The presence of some green staining apparent in the cells reflects loss of membrane integrity which may result from cell death that is independent of extracellular trap release. The experiments performed with M2-HMDMs exposed to interleukin 4 showed no release of DNA from the cells, as indicated by the absence of the strands or streaks of extracellular DNA. However, there was some cellular uptake of green fluorescent dye with the hypochlorous acid and TNF alpha.

It is important to avoid the direct bright light which can overexpose the staining before imaging. Extracellular DNA can be quantified by qPCR analysis on nuclear and mitochondrial DNA present in cell supernatant. Further characterization of mast structure and its roles in chronic inflammation using HMDM may be more clinically relevant in comparison to other immortalized cell line macrophages stores.