Nigericin is a potent microbial toxin and potassium ionophore that can induce pyroptosis. It induces a net decrease in intracellular potassium. This is critical for the oligomerization of the NLRP3 inflammasome and caspase-1 activation in pyroptosis.

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Activation of the NLRP3 inflammasome in myeloid cells follows exposure to pathogen-associated molecular patterns (first signal) and ATP (second signal). This leads to oligomerization and assembly of a high molecular weight (~700 kDa) multimeric inflammasome complex, which leads to the conversion of pro-caspase-1 into the catalytically active form. Inflammatory caspases, such as caspase-1, or interleukin-converting enzyme (ICE), play a central role in innate immunity by recognizing cytosolic foreign danger signals and initiating a two-fold response. First, caspase-1 proteolytically converts the proforms of the two important pro-inflammatory cytokines, interleukin-1ß (IL-1ß) and interleukin-18 (IL-18), into their active forms, which are secreted. Second, caspase-1 or caspase-11 triggers a form of lytic, programmed cell death known as pyroptosis.

Nigericin is a potent microbial toxin derived from Streptomyces hygroscopicus. It acts as a potassium ionophore, inducing a net decrease in intracellular levels of potassium, which is crucial for oligomerization of the NLRP3 inflammasome and activation of caspase-1. Nigericin requires signaling through pannexin-1 to induce caspase-1 activation and IL-1ß processing and release. It has been shown to generate a robust caspase-1 activation response in various cell lines, including Jurkat and THP-I cells.

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  1. Reconstitute each vial of Nigericin with 100 µL DMSO to form the 5 mM stock concentrate. Once reconstituted, it may be aliquoted and stored at ≤ -20°C for 1 year protected from light and thawed no more than twice during that time.
  2. Immediately prior to addition to the samples and controls, dilute 5 mM Nigericin stock 1:10 in diH2O to form a 500 µM working solution for use in treating samples. For example, dilute 1:10 by adding 20 µL stock concentrate to 180 µL diH2O.
  3. Use Nigericin at 1-20 µM to induce NLRP3 inflammasome activation in cells. For example, to use at 10 µM, dilute 500 µM working solution 1:50 in samples; e.g., spike 294 µL cell suspension/overlay medium with 6 µL of 500 µM working solution. Typical treatment periods range from 3-24 hours at 37°C. Each investigator should adjust the concentration of Nigericin and treatment period to accommodate the particular cell line and research conditions.
  4. NLRP3 inflammasome activation can be detected by ELISA or Western blot measuring secreted pro-inflammatory cytokines IL-1β or IL-18, or through the use of caspase-1 activation assays, such as ICT’s Caspase-1 Assay Kits or Pyroptosis/Caspase-1 Assay Kit.

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