AbstractInflammasome is a large protein complex activated upon cellular stress or microbial infection, which triggers maturation of pro-inflammatory cytokines interleukin-1β and interleukin-18 through caspase-1 activation. Nod-like receptor family protein 3 (NLRP3) is the most characterized inflammasome activated by various stimuli. However, the mechanism of its activation is unclear and its exact cellular localization is still unknown. We examined the potential co-localization of NLRP3 inflammasome with mitochondria and seven other organelles under adenosine triphosphate, nigericin or monosodium urate stimulation in mouse peritoneal macrophages using confocal microscopy approach. Our results revealed that the activated endogenous apoptosis-associated speck-like protein containing a CARD (ASC) pyroptosome forms in the cytoplasm and co-localizes with NLRP3 and caspase-1, but not with any of the organelles screened. This study indicates that the ASC pyroptosome universally localizes within the cytoplasm rather than with any specific organelles.