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A Lactate Dehydrogenase Release Assay To Detect Macrophage Death Following Nigericin Exposure

A Lactate Dehydrogenase Release Assay To Detect Macrophage Death Following Nigericin Exposure

筆記録

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.

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