Westers Theresia M, Houtenbos Ilse, Schuurhuis Gerrit Jan, Ossenkoppele Gert J, van de Loosdrecht Arjan A
Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands.
Cytometry A. 2005 Jul;66(1):71-7. doi: 10.1002/cyto.a.20146.
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.
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.
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.
The presented flow cytometric cytotoxicity assay enables the study of T-cell-mediated apoptosis in a heterogenous leukemia population.
树突状细胞向初始T细胞呈递抗原的独特能力在癌症治疗中得到越来越多的应用。基于细胞的免疫疗法的疗效可通过体外测定T细胞对肿瘤细胞的细胞毒性活性来分析。本研究提供了一种流式细胞术方法，用于在单细胞水平分析T细胞对异质性白血病细胞群体的细胞毒性活性。
荧光探针SYTO16和死细胞染料7-氨基放线菌素-D（7-AAD）与白血病细胞识别标记物结合使用，以鉴定凋亡的早期和晚期阶段。通过加入荧光珠来测定活细胞、凋亡细胞和坏死细胞。
在9个急性髓系白血病样本和3个白血病细胞系中，与单独使用7-AAD相比，SYTO16与死细胞标记物7-AAD联合使用显著提高了细胞毒性测定的灵敏度（P = 0.001）。对几个效应细胞与靶细胞比例的分析揭示了确定剂量反应效应的能力。绝对数量的计数结果显示，细胞系和白血病样本的变异系数分别为4.1%和8.4%。
所提出的流式细胞术细胞毒性测定法能够研究异质性白血病群体中T细胞介导的凋亡。
