Quantification of T-cell-mediated apoptosis in heterogeneous leukemia populations using four-color multiparameter flow cytometry

Theresia M. Westers, Ilse Houtenbos, Gerrit Jan Schuurhuis, Gert J. Ossenkoppele, Arjan A. Van De Loosdrecht*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review


Background: The unique capacity of dendritic cells to present antigens to naive T cells is being increasingly utilized in cancer therapy. The efficacy of cell-based immunotherapy can be analyzed by determination of cytotoxic activity of T cells toward tumor cells in vitro. This study supplies a flow cytometric method to analyze T-cell-mediated cytotoxic activity toward heterogeneous leukemic cell populations at a single-cell level. Methods: The fluorescent probe SYTO16 and the dead-cell dye 7-aminoactinomycine-D (7-AAD) were used to identify early and late stages of apoptosis in combination with leukemia cell-identifying markers. Determination of viable, apoptotic, and necrotic cells was performed by inclusion of fluorescent beads. Results: In nine acute myeloid leukemia samples and three leukemic cell lines the use of SYTO16 next to the dead-cell marker 7-AAD significantly increased (P = 0.001) the sensitivity of the cytotoxicity assay as compared with single use of 7-AAD. Analysis of several effector-to-target ratios revealed the ability to determine dose-response effects. Enumeration of absolute numbers resulted in coefficients of variation of 4.1% and 8.4% for cell lines and leukemic samples, respectively. Conclusion: The presented flow cytometric cytotoxicity assay enables the study of T-cell-mediated apoptosis in a heterogenous leukemia population.

Original languageEnglish
Pages (from-to)71-77
Number of pages7
JournalCytometry Part A
Issue number1
Publication statusPublished - Jul 2005

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