Simultaneous Detection of Inflammasome Activation and Membrane Damage During Pyroptosis

Pyroptosis is a highly regulated inflammatory form of cell death that plays a role in many different diseases, including cancer. Pyroptosis was initially described to be mediated by caspase-1, which is activated by innate immune signaling complexes called inflammasomes. Inflammasomes trigger caspase-dependent activation of the pore-forming protein, gasdermin D, and plasma membrane disruption. In this protocol, we describe a method to simultaneously detect two hallmarks of inflammasome-mediated pyroptosis. Using a fluorescently tagged inflammasome adaptor protein (ASC-Citrine) and membrane-impermeable nuclear dyes, we can track inflammasome formation and plasma membrane disruption over time in the same cell population.