Measuring NLR Oligomerization II: Detection of ASC Speck Formation by Confocal Microscopy and Immunofluorescence

Abstract

Inflammasome assembly results in the formation of a large intracellular protein scaffold driven by the oligomerization of the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). Following inflammasome activation, ASC polymerizes to form a large singular structure termed the ASC "speck," which is crucial for recruitment of caspase-1 and its inflammatory activity. Hence, due to the considerably large size of these structures, ASC specks can be easily visualized by microscopy as a simple upstream readout for inflammasome activation. Here, we provide two detailed protocols for imaging ASC specks: by (1) live-cell imaging of monocyte/macrophage cell lines expressing a fluorescently tagged version of ASC and (2) immunofluorescence of endogenous ASC in cell lines and human immune cells. In addition, we outline a protocol for increasing the specificity of ASC antibodies for use in immunofluorescence.

Keywords: ASC; Confocal microscopy; Flow cytometry; Immunofluorescence; Inflammasome; Live-cell imaging; Speck.