Encyclopedia of Experiments: Immunology
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Transcript
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.
