Cell-Mediated Cytotoxicity Evaluation Using Monoclonal Antibody Staining for Target or Effector Cells with AnnexinV/Propidium Iodide Colabeling by Fluorosphere-Adjusted Counts on Three-Color Flow Cytometry

Background: In addition to ⁵¹chromium release assay, flow cytometric methods have been described to assess in vitro cell-mediated cytotoxicity. In this report, we describe a new flow cytometric approach for determination of in vitro cell-mediated cytotoxicity utilizing three-color flow cytometric assay. Methods: This method is based on monoclonal antibody staining of either effector or target cells to evaluate cytotoxicity with increased accuracy by utilizing fluorospheres for calibration. The basic strategy involves labeling effector or target cells with a specific fluorescent-conjugated monoclonal antibody, in addition to staining with annexinV-FITC and propidium iodide to identify apoptotic/dead cells. The effector and target cell populations as well as conjugates were clearly and easily identified by this approach. Results: We obtained significant correlation between cytotoxicity calculated by this technique and ⁵¹chromium release assay results. The integration of fluorospheres allowed us to determine the absolute number of events reflective of the cumulative cell death rather than a crosssectional, percentage-based cytotoxicity assessment in the target cell population at the time of analysis. Conclusions: This method provides additional advantages to other methods and enables the study of target cell fate in more detail, as well as providing a potential contribution to understanding the mechanisms of cell elimination.