ASC speck formation as a readout for inflammasome activation

Methods Mol Biol. 2013;1040:91-101. doi: 10.1007/978-1-62703-523-1_8.

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.

MeSH terms

  • Animals
  • Apoptosis Regulatory Proteins
  • CARD Signaling Adaptor Proteins
  • Cell Line
  • Cytoskeletal Proteins / genetics
  • Cytoskeletal Proteins / metabolism*
  • Gene Expression
  • Genetic Vectors / genetics
  • Humans
  • Inflammasomes / immunology
  • Inflammasomes / metabolism*
  • Macrophages / immunology
  • Macrophages / metabolism
  • Mice
  • Microscopy, Confocal
  • Retroviridae / genetics
  • Transduction, Genetic

Substances

  • Apoptosis Regulatory Proteins
  • CARD Signaling Adaptor Proteins
  • Cytoskeletal Proteins
  • Inflammasomes
  • Pycard protein, mouse