However, the crucial experiment of incubating islets lacking inflammasome components or IL-1 receptors with the toxic stimulus was not done. When we performed this experiment, we found that loss of functional inflammasome factors, NLRP3 or caspase-1, did not inhibit glucose toxicity, ER stress or oxidative stress-mediated death of islet cells. These findings agree with previous data demonstrating that islets lacking IL-1 receptors are not protected from glucose toxicity. Consistent with our results, another study showed that treatment of isolated human islets with high glucose concentration for up to 7 days neither induced IL-1b secretion nor affected IL-1b gene expression. Our data are also in agreement with the concept that mouse islets are not susceptible to killing by IL-1b on its own, but require the addition of IL-1b+IFNc to induce significant cell death. Nevertheless, differences in the source of islets, the number of islets, culture conditions, drug concentrations, differences between various beta-cell lines and assays used to measure cell death and inflammasome activity can not be ruled out as possible explanations for the differences between our data and results from some of the previous studies. For example, Maedler and colleagues cultured human islets for 2 days on extracellular matrix coated plates allowing the cells to attach and spread before treating islets with glucose that led to IL-1b secretion. On the other hand we, and others, cultured islets in noncoated petri dishes and treated them with various reagents after 24 hours of isolation. The DNA fragmentation assay we used in our experiments has the advantage that we analyze 10,000 cells per sample, which makes this a highly sensitive method for picking up subtle differences in treatments or genotypes. Although trypsinizing cells does induce some cell death, the proportion of dead cells as a result of trypsinizing was consistent across all treatment groups. Nevertheless, we cannot rule out the possibility that small increases in islet cell death induced by IL-1b could have been masked by cell death induced by trypsinization. Another caveat is the use of LPS in our study to induce signal 1 in islets, where it is possible that other agents, such as SAR131675 Pam2CSK4 may provide a better signal. The story may be further complicated during diabetes in vivo during which macrophages recuited to the islet could increase IL1b production. Compared to macrophages, the expression of NLRP3 inflammasome components is very low in islet beta cells, and beta cells produced only a modest amount of IL-1b after glucose treatment. Even when we used islets with beta-cell specific expression of a NLRP3 activating mutation, IL-1b production was almost undetectable and glucose toxicity remained similar to wildtype islets. While islet resident macrophages may be a potential source of islet IL-1b, deficiency of either NLRP3 or caspase-1, both components of the NLRP3 inflammasome required for IL-1b processing in macrophages, did not affect in vitro glucose toxicity mediated killing of whole islets, suggesting the contribution of resident macrophage IL-1b is not significant in this context.