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Three-color flow cytometric assay for the study of the mechanisms of cell-mediated cytotoxicity

  1. Author:
    Derby, E.
    Reddy, V.
    Kopp, W.
    Nelson, E.
    Baseler, M.
    Sayers, T.
    Malyguine, A.

  2. Author Address

    NCI, SAIC, POB B, Frederick, MD 21702 USA. Univ Calif Irvine, Irvine, CA 92697 USA. NCI, SAIC, Frederick, MD 20878 USA. Malyguine A NCI, SAIC, POB B, Frederick, MD 21702 USA.
    1. Year: 2001
  2. Journal: Immunology Letters
    1. 78
    2. 1
    3. Pages: 35-39
  4. Type of Article: Article
  1. Abstract:

    Cytotoxic lymphocytes kill tumor or virus-infected target cells utilizing two mechanisms (1) release of lytic granules (containing perform and granzymes) and (2) Fas ligand (FasL)/Fas or TNF initiated apoptosis. We have examined mechanisms of target cell lysis using a new Flow Cytometric Cytotoxicity Assay (FC Assay). Target cells were labeled with PKH 67 dye. Cell death was estimated by 7-amino-actinomycin (7- AAD) inclusion and annexin V-PE binding. A strong direct correlation has been found between the percentage of dead target cells in the FC Assay and the results of Cr-51 release assay when human LAK and CTL were used as a model system. We have shown that both NK and CTL kill tumor cells mostly by granule-mediated mechanisms, as lysis was blocked by a perforin inhibitor Concanamycin A (Folimycin) but was significantly less sensitive to zVAD-FMK caspase inhibition. The FC assay allows accurate measurement of cell-mediated cytotoxicity as individual target cell death is detected directly. (C) 2001 Elsevier Science BN. All rights reserved.

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