Ž . Journal of Immunological Methods 258 2001 183–191 www.elsevier.comrlocaterjim Protocol Antibody-dependent cell-mediated cytotoxicity: a flow cytometry-based assay using fluorophores Robert W. Wilkinson a,1,2 , Alice E. Lee-MacAry a, ),1 , Derek Davies b , David Snary a , Elizabeth L. Ross a a ( ) Applied DeÕelopment Laboratory, Imperial Cancer Research Technology ICRT , Dominion House, St Bartholomew’s Hospital, London EC1A 7BE, UK b ( ) FACS Laboratory, Imperial Cancer Research Fund ICRF , London WC2A 3PX, UK Received 5 July 2001; accepted 25 July 2001 Abstract Ž . Using flow cytometry FCM , an assay has been developed for the determination of antibody-dependent cell-mediated Ž . cytotoxicity ADCC . Target cells were labelled with a membrane dye, PKH-26, to allow discrimination when incubated with effector cells and antibody. Post-incubation, cell death within the PKH-26 q target cell population was assessed by the Ž . addition of the viability probe TO-PRO-3 iodide TP3 . This ADCC method allows analysis to be conducted on a single cell basis and overcomes the need for radiochemicals. This communication indicates that the assay is accurate and reproducible with the potential to be a useful tool for evaluating the therapeutic potential of antibodies and antibody-based reagents. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Flow cytometry; Antibody-dependent cell-mediated cytotoxicity; PKH-26; TO-PRO-3 iodide 1. Type of research Antibodies are increasingly emerging as useful adjuncts to therapy for a number of diseases includ- Ž . ing cancer and autoimmunity Vaughan et al., 1998 . AbbreÕiations: ADCC, antibody-dependent cell-mediated cy- totoxicity; FCM, flow cytometry; CM, complete medium; FCS, foetal calf serum; TP3, TO-PRO-3 iodide. ) Corresponding author. Tel.:q 44-020-7726-4708; fax: q 44- 020-7796-3907. E-mail address: A.Lee-MacAry@icrf.icnet.uk Ž . A.E. Lee-MacAry . 1 Contributed equally to the work. 2 Present Address: Department of Cancer and Infection Re- search, AstraZeneca Pharmaceuticals, Macclesfield, Cheshire, UK. In vivo antibodies mediate their effect directly via activation or inhibition of biochemical pathways or indirectly via recruitment of complement serum pro- teins or effector immune cells such as natural killer Ž . Ž . NK cells Cragg et al., 1999 . Effector cells destroy their target by recognising surface-bound immuno- Ž . Ž . globulin Ig via IgG–Fc region receptors Fc gR through a mechanism known as antibody-dependent Ž .Ž . cell-mediated cytotoxicity ADCC Perussia, 1998 . At present, many ADCC assays use the radioiso- 51 Ž 51 .Ž . tope Chromium Cr Brunner et al., 1968 . Al- though widely used 51 Cr-based assays have several drawbacks: 51 Cr is expensive and volatile; there may be high spontaneous release of the probe; certain cell types are difficult to load; and finally, radioisotopes may have a detrimental effect upon cell function. We 0022-1759r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. Ž . PII: S0022-1759 01 00474-4