Research paper A new flow cytometric assay for the simultaneous analysis of antigen-specific elimination of T cells in heterogenous T cell populations Christian Schütz a, ⁎, Karin Fischer b , Simon Völkl c , Sabine Hoves d , Dagmar Halbritter a , Andreas Mackensen c , Martin Fleck a a University of Regensburg, Department of Internal Medicine I, Franz-Josef-Strauss-Allee 11, 93042 Regensburg, Germany b University of Regensburg, Department of Internal Medicine I, Division of Hematology and Oncology, 93042 Regensburg, Germany c University of Erlangen-Nuernberg, Department of Internal Medicine V, 91054 Erlangen, Germany d Peter MacCallum Cancer Centre, East Melbourne, VIC 3002, Australia article info abstract Article history: Received 20 November 2008 Received in revised form 11 March 2009 Accepted 11 March 2009 Available online 28 March 2009 Novel immunosuppressive strategies are targeting for an antigen-specific deletion of T cells responsible for organ damage in autoimmunity and allograft rejection. Here, we present a new flow cytometry-based assay that allows the reliable and efficient detection of T cells that were eliminated in an antigen-specific fashion. A stable cell-labelling technique utilizing the two membrane dyes PKH26 and PKH67 has been combined with annexin V and 7-aminoactinomycin (7-AAD) staining to detect apoptotic cells. A differential gating strategy enabled us to determine the viability/apoptosis for each PKH-stained T cell subpopulation independently. The capability to simultaneously analyze apoptosis within T cell mixtures of different antigen specificities establishes this assay as a superior tool for the further development of novel antigen-specific immunosuppressive approaches. © 2009 Elsevier B.V. All rights reserved. Keywords: Flow cytometry PKH26 PKH67 Cytotoxic T lymphocytes Apoptosis 1. Introduction Several methods have been established to quantify and characterize autoreactive T cells, which have been applied to numerous in vitro and in vivo models. Based on the results obtained from these studies novel therapeutic strategies aim to delete autoreactive T cells in an antigen-specific fashion in order to minimize general immunosuppression, which is the major side effect of current undirected treatments. However, assays that determine the elimination of antigen-specific T cells with- in a mixture of T cells with different antigen specificities are lacking. Here, we describe a new flow cytometric assay that allows for the quantification of viable as well as apoptotic T cell subpopulations with different antigen specificities in one sample, simultaneously. So far, cell viability has been analyzed utilizing different methods based on the release of radioactive tracers from lyzed cells. In general, these reagents are compounds containing radioactive isotopes like 51 Chromium ( 51 Cr), 75 Selenium ( 75 Se) or Tritium ( 3 H) (Cerottini and Brunner, 1974; Mantovani et al., 1979; Hoves et al., 2003). As the usage of a radioactive material leads to various potential hazards, several non-radioactive assays were developed based on the labelling with markers such as Europium (Eu 3+ )(Patel and Boyd, 1995), bisbenzamid dye (Toka et al., 1996) or Calcein-AM (Lichtenfels et al., 1994). However, all these assays are dependent on the release of the compounds to the supernatant during cell death, which has the drawback of low accuracy. Novel assays for the assessment of cell death utilize flow cytometry (Johann et al., 1995; Mattis et al., 1997; Aubry et al., 1999; Flieger et al., 1999; Fischer et al., 2002; Fischer and Mackensen, 2003). As a major advantage, these assays provide a high sensitivity on the single-cell level as well as the possibility to additionally perform phenotypic analyses. The method described in this article uses the two fluorescent membrane dyes PKH26 (red) and PKH67 (green) to trace T cell subpopulations bearing different antigen specificities in a T cell mixture of various antigen specificities. Journal of Immunological Methods 344 (2009) 98–108 ⁎ Corresponding author. Tel.: +49 941944 7170; fax: +49 941 944 7123. E-mail address: christian.schuetz@klinik.uni-r.de (C. Schütz). 0022-1759/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2009.03.008 Contents lists available at ScienceDirect Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim