This article outlines a protocol for assessing pyroptosis in macrophages using an LDH release assay. The method involves priming macrophages with lipopolysaccharide and subsequently treating them with nigericin to trigger NLRP3 inflammasome activation.
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.
To perform an LDH release assay, add 50 microliters of DMEM-5 medium supplemented with 10 micromolar nigericin to each experimental well, and 50 microliters of DMEM-5 to the spontaneous, and 100% lysis control wells for 60 minutes at 37 degrees Celsius and 5% carbon dioxide. After 30 minutes add 10 microliters of 10x lysis buffer to the 100% lysis control wells, and add 10 microliters of medium to the other wells. At the end of the incubation centrifuge the plate, and transfer 50 microliters of supernatant from each well to a clear flat-bottom plate.
Next add 50 microliters of freshly thawed and prepared substrate to each well for a 30-minute incubation in the dark, checking the plate after 15 minutes to confirm that the signal will not exceed the detection limit of the plate reader. At the end of the incubation, add 50 microliters of stop solution to each well, and measure the OD490 on an appropriate plate reader to allow calculation of the percentage of cell lysis per well.
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