Recombinant Mouse CCL3

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.