Encyclopedia of Experiments: Immunology
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Transcript
Take an assay plate containing macrophages primed with lipopolysaccharide.
Priming increases the expression of inactive pro-inflammatory cytokine precursors and NLRP3 proteins.
Add nigericin, a potassium ionophore, promoting ion efflux. This disrupts intracellular potassium levels, triggering NLRP3 proteins, to form a large complex.
Oligomerized NLRP3 recruits the adaptor proteins, ASC, causing their polymerization. ASC filaments recruit pro-caspase-1, forming NLRP3-inflammasome, a multiprotein complex.
Pro-caspase-1 undergoes proximity-induced auto-activation, generating caspase-1. Caspase-1 cleaves the pro-inflammatory cytokine and gasdermin-D protein, creating a plasma membrane pore.
This results in cytokine secretion and inflammatory programmed cell death, pyroptosis, releasing intracellular contents, including lactate dehydrogenase, or LDH. Collect the LDH-containing supernatant.
Add substrate mixture — lactate, iodonitrotetrazolium violet, or INT dye, NAD+, and diaphorase. LDH oxidizes lactate to pyruvate while reducing NAD+. Diaphorase catalyzes the reduction of INT, forming red-colored formazan.
The color intensity corresponds to LDH release, with elevated levels indicating increased pyroptosis-associated cell damage and death.
