This article describes a protocol for studying the activation of the NLRP3 inflammasome in primary macrophages. The process involves the introduction of bacterial lipopolysaccharide and subsequent treatments to visualize inflammasome formation and pyroptosis using fluorescence microscopy.
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.
For immunofluorochemical analysis of the nigericin-treated cells, wash the probe-labeled cells, and incubate the cells in 250 microliters of fixation and permeabilization solution per well for 30 minutes on ice, protected from light.
Wash the macrophages three more times with fresh wash buffer, and label the cells with 250 microliters of primary antibody against apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain, or ASC, per well for one hour on ice.
Wash the cells at the end of the incubation, and label the samples with 250 microliters of the appropriate fluorescent dye-conjugated secondary antibody for another hour on ice, adding 4 microliters of an appropriate fluorescent nuclear staining dye during the last 5 minutes.
After washing the cells 3 times in cold wash buffer, mount coverslips onto the slides with 7 microliters of anti-fade mounting medium as demonstrated, and image the macrophages by fluorescence confocal microscopy.
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