Development of a novel flow cytometric cell-mediated cytotoxicity assay using the fluorophores PKH-26 and TO-PRO-3 iodide

Alice E. Lee-MacAry, Elizabeth L. Ross, Derek Davies, Ruthline Laylor, Jamie Honeychurch, Martin J. Glennie, David Snary, Robert W. Wilkinson

    Research output: Contribution to journalArticlepeer-review

    Abstract

    A flow cytometric (FCM) assay has been developed for the determination of cell-mediated cytotoxicity (CMC). In the assay, the target tumour cell population was labelled with a membrane dye, PKH-26, prior to incubation with splenocyte effector cells. Cell death within the target population was assessed by the addition of the viability probe TO-PRO-3 iodide (TP3) and analysed by flow cytometry. The extent of cytotoxicity was determined by the relative number of live target cells labelled with PKH-26 only and dead, permeabilised cells labelled with both PKH-26 and TP3. This CMC method allows the analysis to be conducted on a single cell basis and overcomes the need for radiochemicals. This communication indicates that the FCM assay is an accurate and reproducible experimental system capable of analysing natural killer (NK) cell and antibody-dependent cell-mediated cytotoxicity. The procedure is comparable to the chromium release assay. We believe that this is one of the first demonstrations of an FCM-based antibody-dependent cell-mediated cytotoxicity (ADCC) assay. © Elsevier Science B.V.
    Original languageEnglish
    Pages (from-to)83-92
    Number of pages9
    JournalJournal of immunological methods
    Volume252
    Issue number1-2
    DOIs
    Publication statusPublished - 1 Jun 2001

    Keywords

    • Cell-mediated cytotoxicity
    • Flow cytometry
    • PKH-26
    • TO-PRO-3 iodide

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