ASC Speck Formation as a Readout for Inflammasome Activation

Abstract

All inflammasomes require the adapter protein apoptosis associated speck-like protein containing a CARD (ASC) for the activation of caspase-1. After inflammasome activation, ASC assembles into a large protein complex, which is termed "speck". ASC specks can be observed as they reach a size of around 1 μm and in most cells only one speck forms upon inflammasome activation. Hence, ASC speck formation can be used as a simple upstream readout for inflammasome activation. Here, we describe a method for analyzing inflammasome activation by ASC speck visualization. First, we describe the generation of a clonal inflammasome reporter macrophage cell line overexpressing fluorescently tagged ASC. We then discuss stimulation conditions and the microscopic evaluation of ASC speck formation.