Visualization and quantification of T cell-mediated cytotoxicity using cell-permeable fluorogenic caspase substrates

Nat Med. 2002 Feb;8(2):185-9. doi: 10.1038/nm0202-185.


We have developed a non-radioactive flow-cytometry assay to monitor and quantify the target-cell killing activities mediated by cytotoxic T lymphocytes (CTLs). This flow-cytometry CTL (FCC) assay is predicated on measurement of CTL-induced caspase activation in target cells through detection of the specific cleavage of fluorogenic caspase substrates. Here we show that this assay reliably detects antigen-specific CTL killing of target cells, and demonstrate that it provides a more sensitive, more informative and safer alternative to the standard 51Cr-release assay most often used to quantify CTL responses. The FCC assay can be used to study CTL-mediated killing of primary host target cells of different cell lineages, and enables the study of antigen-specific cellular immune responses in real time at the single-cell level. As such, the FCC assay can provide a valuable tool for studies of infectious disease pathogenesis and development of new vaccines and immunotherapies.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Caspases / metabolism*
  • Chromium Radioisotopes
  • Cytotoxicity, Immunologic / physiology*
  • Female
  • Flow Cytometry / methods
  • Mice
  • Mice, Inbred C57BL
  • Substrate Specificity
  • T-Lymphocytes, Cytotoxic / immunology*


  • Chromium Radioisotopes
  • Caspases