Nigericin is a potent microbial toxin that acts as a potassium ionophore, inducing a net decrease in intracellular levels of potassium. This is critical for the oligomerization of the NLRP3 inflammasome and activation of caspase-1 in pyroptosis. Nigericin can be used as a positive control in pyroptosis experiments.
- 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.
- 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.
- 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.
- 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.