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. (c) 2005 Wiley-Liss, Inc.
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. (c) 2005 Wiley-Liss, Inc.
Authors: Martine E D Chamuleau; Theresia M Westers; Linda van Dreunen; Judith Groenland; Adri Zevenbergen; Corien M Eeltink; Gert J Ossenkoppele; Arjan A van de Loosdrecht Journal: Haematologica Date: 2009-02-19 Impact factor: 9.941
Authors: Willemijn van den Ancker; Marvin M van Luijn; Jurjen M Ruben; Theresia M Westers; Hetty J Bontkes; Gert J Ossenkoppele; Tanja D de Gruijl; Arjan A van de Loosdrecht Journal: Cancer Immunol Immunother Date: 2010-09-22 Impact factor: 6.968
Authors: Willemijn van den Ancker; Jurjen M Ruben; Theresia M Westers; Dewi Wulandari; Hetty J Bontkes; Erik Hooijberg; Anita G M Stam; Saskia J A M Santegoets; Gert J Ossenkoppele; Tanja de Gruijl; Arjan van de Loosdrecht Journal: Oncoimmunology Date: 2013-04-01 Impact factor: 8.110