Visualization of Extracellular Traps in Macrophages from Human Bronchoalveolar Lavage Fluids

During infection, alveolar macrophages release macrophage extracellular traps or METs to eliminate pathogens.

METs consist of chromatin fibers complexed with peptidyl arginine deiminases, PADs, matrix metalloproteinases,  and citrullinated histones.

To visualize METs in macrophages from bronchoalveolar lavage fluid, first, centrifuge the fluid.

Plate the resuspended cells on coverslips within multi-well plate wells.

Macrophages attach to the coverslips, while other cells remain suspended and are discarded.

Fix the macrophages, preserving the cellular structure. Permeabilize them to access the cellular targets.

Add blocking proteins, preventing non-specific antibody binding.

Supplement with a primary antibody cocktail that binds to target citrullinated histones, PADs, and matrix metalloproteinases, respectively.

Add different fluorophore-tagged secondary antibodies that bind to target MET protein-bound primary antibodies.

Mount the cells with DAPI-containing mounting medium. DAPI, a fluorescent dye, stains the chromatin fibers.

Using a confocal microscope, visualize the METs characterized by fluorescent extracellular chromatin fibers with citrullinated histones, PADs, and metalloproteinases.

After obtaining lung macrophages from humans and mice by bronchoalveolar lavage, or BAL, according to the text protocol, use trypan blue and a hemocytometer to perform a viable cell count on the BAL solution. Pipette 1 to 4 x 10⁵ macrophages in 500 microliters of culture medium onto coverslips in the wells of 24-well plates. Then, incubate the cells at 37 degrees Celsius overnight for the cells to adhere.

Remove the culture medium and use PBS to wash the cells once. Then, add 2% paraformaldehyde/periodate/lysine or PLP fixative, and incubate the samples for 10 minutes. After using PBS to briefly wash the cells, add 0.2% Tween-20 in PBS, and incubate the cells for 20 minutes.

Next, to block the cells, add 10% chicken serum in 5% BSA diluted in PBS, and incubate the samples at room temperature for 30 minutes. Then, replace the block solution with a 1 to 100 concentration of primary and isotope control antibodies, and incubate the cells at room temperature for one hour. Following the incubation, use PBS to wash the cells before adding the corresponding secondary antibodies. Then, incubate the samples for 40 minutes at room temperature.

After washing the cells in PBS, mount the samples with DAPI-based mounting medium. Then, visualize the samples using a confocal microscope.