Imaging-based methods for assessing caspase activity in single cells

Cold Spring Harb Protoc. 2015 Jan 5;2015(1):pdb.top070342. doi: 10.1101/pdb.top070342.


Caspases, a family of proteases that are essential mediators of apoptosis, are divided into two groups: initiator caspases and executioner caspases. Each initiator caspase is activated at the apex of its respective pathway, which generally leads to the cleavage and activation of executioner caspases. Executioner caspases in turn cleave numerous substrates in the cell, leading to its demise. Initiator caspases are activated when inactive monomers undergo induced proximity to form an active caspase. In contrast, executioner caspases are activated by cleavage. Based on this key difference, different imaging techniques have been developed to measure caspase activation and activity on a single-cell basis. Bimolecular fluorescence complementation (BiFC) is used to measure induced proximity of initiator caspases, whereas Förster resonance energy transfer (FRET) permits the investigation of caspase-mediated substrate cleavage in real time. Because many of the events in apoptosis, including caspase activation, are asynchronous in nature, these single-cell imaging techniques have proven to be immensely powerful in ordering and dissecting caspase pathways. When coupled with parallel detection of additional hallmark events of apoptosis, they provide detailed and quantitative kinetic and positional insights into the signal transduction pathways that regulate cell death. Here we provide a brief introduction into BiFC- and FRET-based imaging of caspase activation and activity in single cells.

MeSH terms

  • Animals
  • Apoptosis*
  • Caspases / metabolism*
  • Cells, Cultured
  • Enzyme Activation / drug effects
  • Fluorescence Resonance Energy Transfer
  • Humans
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Models, Molecular
  • Peptides / pharmacology
  • Transfection


  • Luminescent Proteins
  • Peptides
  • TcapQ647 peptide
  • Caspases