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

A flow cytometric (FCM) assay has been developed for the determination of c ell-mediated cytotoxicity (CMC). In the assay, the target tumour cell popul ation was labelled with a membrane dye, PKH-26, prior to incubation with sp lenocyte effector cells. Cell death within the target population was assess ed by the addition of the viability probe TO-PRO-3 iodide (TP3) and analyse d by flow cytometry. The extent of cytotoxicity was determined by the relat ive number of live target cells labelled with PKH-26 only and dead, permeab ilised 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 FCR;I assay is an acc urate and reproducible experimental system capable of analysing natural kil ler (NK) cell and antibody-dependent cell-mediated cytotoxicity. The proced ure is comparable to the chromium release assay. We believe that this is on e of the first demonstrations of an FCM-based antibody-dependent cell-media ted cytotoxicity (ADCC) assay, (C) 2001 Elsevier Science B.V. All rights re served.