Rapid flow cytometry-based assay for the evaluation of γδ T cell-mediated cytotoxicity.

ABSTRACT

The effector function of natural killer, lymphokine--activated killer cells and T lymphocytes is commonly evaluated by radioactive chromium­release cytotoxicity assays. In addition to this indirect method, fluorescence assays have been described for the assessment of in vitro cell­mediated cytotoxicity. In the present study, target cells were stained with 5­(and­6)­carboxyfluorescein diacetate succinimidyl ester (CFSE), which is a stable integrated fluorescent probe that allows target and effector cells to be distinguished from one another. Staining of target THP­1 cells with 8 µM CFSE revealed high and stable loading of fluorescence and no effect of the viability of cells. After 4 h of in vitro co­culture between γδ T cells and CFSE­labeled infected or uninfected THP­1 cells, staining with propidium iodide (PI) was performed to distinguish between vital and dead cells. During sample acquisition, target cells were gated on the CFSE positivity and examined for cell death based on the uptake of PI. CFSE and PI double positive cells were recognized as the dead target cells. The percentage of cytotoxicity in the CFSE­gated cell population was calculated by subtracting the value obtained for non­specific PI­positive target cells, which was measured in a control group that did not contain effector cells. The present study describes a simple and convenient assay that is based on the direct quantitative and qualitative analysis of cell damage at a single cell level utilizing a two­color flow cytometric assay. In conclusion, the flow cytometric­based assay described in the current study is a simple, sensitive and reliable tool to determine the cytolytic activity of γδ T lymphocytes against mycobacteria. Therefore, the present study may provide valuable information concerning the methods employed to investigate the function of γδ T cells and potentially other lymphocyte subsets.