A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells:: Comparisons to a 4 h 51Cr-release assay
A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells:: Comparisons to a 4 h 51Cr-release assay
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DOI：
10.1016/j.jim.2007.05.013
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发表时间：
2007-08-31
影响因子：
2.2
通讯作者：
Whiteside, T. L.
中科院分区：
文献类型：
--
作者：
Kim, G. G.;Donnenberg, V. S.;Whiteside, T. L.
自然杀伤（NK）细胞或T细胞介导的细胞毒性传统上在4-16小时Cr-51释放测定（CRA）中测量。建立了一种新的基于四色流式细胞术的细胞毒试验（FCC），可同时测定NK细胞的细胞毒活性和NK细胞表型（CD 3（-）CD 16（+）CD 56（+））。在加入效应细胞之前，用Cell Tracker橙子（CTO）标记靶细胞K562或Daudi。共孵育后，加入7-氨基-放线菌素D（7-AAD）以测量靶细胞的死亡。基于光散射（FSC/SSC）、荧光峰积分和高度的双重辨别以及荧光强度，测量效应物的表型、靶的活力、肿瘤-效应细胞缀合物的形成以及所有细胞的绝对数量。在不同效应细胞与靶细胞（E：T）比例（50、25、12和6）下进行的动力学研究（0.5和1 - 4 h）证实，孵育3 h是最佳的。FCC测定比CRA更灵敏，具有8-13%的变异系数（CV），并且可靠地测量正常对照和癌症受试者中NK细胞或淋巴因子激活的杀伤（LAK）细胞介导的靶细胞杀伤。FCC测定可用于研究一系列表型属性，除了效应细胞的各种亚群的裂解活性之外，而无需放射性示踪剂，因此相对便宜。FCC测定具有提供关于靶细胞裂解的分子相互作用的信息的潜力，从而成为研究疾病发病机制以及开发新型免疫疗法的主要工具。(C)2007 Elsevier B. V.保留所有权利。
Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h Cr-51-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies. (C) 2007 Elsevier B.V. All rights reserved.