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 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.
Lee-MacAry
A E
AE
Applied Development Laboratory, Imperial Cancer Research Technology (ICRT), Dominion House, St. Bartholomew's Hospital, EC1A 7BE, London, UK.
Ross
E L
EL
Davies
D
D
Laylor
R
R
Honeychurch
J
J
Glennie
M J
MJ
Snary
D
D
Wilkinson
R W
RW
D000818:Animals; D000920:Antibody-Dependent Cell Cytotoxicity / Q000276:immunology*; D002232:Carbocyanines; D016923:Cell Death; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D007694:Killer Cells, Natural / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009930:Organic Chemicals; D014407:Tumor Cells, Cultured
D002232:Carbocyanines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C070080:PKH 26; C098830:TO-PRO-3