Characterization of the flow cytometric assay for ex vivo monitoring of cytotoxicity mediated by antigen-specific cytotoxic T lymphocytes.

Several non-radioactive methods have widely been utilized to detect antigen-specific cytotoxic T lymphocyte (CTL) responses instead of the classical 51Cr-release assay. These methods include intracellular cytokine staining, major histocompatibility complex-class I tetramers, and the CD107a mobilization assay. However, they do not directly measure target-cell death. In contrast, several attempts have been made to develop the flow cytometric CTL (FC-CTL) assay for evaluation of cytotoxicity. However, further improvement is necessary for it to become standardized. Here, we evaluated the characteristics of the FC-CTL assay based on the uptake of propidium iodide (PI) using target cell lines expressing the green fluorescent protein (GFP). The FC-CTL assay was found to be sensitive enough to detect primary CTL responses. The usage of a pre-established GFP-expressing target cell line facilitated the procedure of the assay, and enabled a clear discrimination between target and effector cells. Time-course analyses demonstrated that PI-stained target cells were detected as early as surface CD107a expression after antigenic stimulation. Thus, the PI/GFP-based FC-CTL assay is sufficiently sensitive to practically detect the early stages of target-cell death, and may have a great potential for becoming a standard tool to measure CTL activity.