An In Vitro Technique to Study Inflammasome Complex Formation in Macrophages
Transcrição
Begin with a plate containing a culture of primary macrophages.
Introduce bacterial lipopolysaccharide, LPS, that binds to toll-like receptors on macrophages, triggering upregulation of the protein NLR family pyrin domain-containing 3 or NLRP3.
Add an ionophore that mediates the efflux of potassium ions. Decreased cytosolic potassium activates NLRP3. Activated NLRP3 oligomerizes and induces the recruitment of an adaptor protein, which in turn recruits pro-caspase-1, forming an inflammasome.
Auto-proteolysis converts pro-caspase-1 to active caspase-1, which mediates inflammatory cell death or pyroptosis. DNA damage during pyroptosis leads to nuclear condensation.
Fix the cells and treat with a detergent to permeabilize them.
Add a primary antibody binding to the inflammasome-associated adaptor protein, and a fluorescently-labeled secondary antibody that binds to the primary antibody, labeling the inflammasome.
Introduce a fluorescent nucleic acid stain to label the nucleus. Under a fluorescence microscope, cells display condensed nuclei — indicating pyroptosis — and foci of adaptor proteins — indicating inflammasome formation.
