Inflammatory caspases, such as caspase-1, play a critical role in the mechanism to trigger the lytic programmed cell death known as pyroptosis. Nigericin can be utilized as a positive control in pyroptosis experiments.
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. It 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.
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.
ICT’s caspase-1 inhibitor reagent, FAM-YVAD-FMK (kit catalog #9146) was used to monitor the caspase-1 induction response in Jurkat cells treated with Nigericin for various periods of time. A common cell pool was spiked with FAM-YVAD-FMK and divided into separate treatment groups. Starting with 24 hour samples and working backwards, 10 μM Nigericin was added to cells and the samples were incubated at 37°C throughout the induction process. Following their respective treatment exposure periods, the cells were washed and analyzed on an Accuri C6 flow cytometer. The amount of caspase-1 activity detected directly correlated to the duration of the exposure period; the longer the cells were exposed to Nigericin, the larger the proportion of caspase-1 positive cells found in the sample. Data courtesy of Mrs. Tracy Murphy, ICT (220:78).
Molecular weight: 746.94 g/mol
Storage: ≤ -20°C