A novel one-step, highly sensitive fluorometric assay to evaluate cell-mediated cytotoxicity
A novel one-step, highly sensitive fluorometric assay to evaluate cell-mediated cytotoxicity
复制标题
DOI：
10.1016/s0022-1759(98)00028-3
复制
发表时间：
1998-04-15
影响因子：
2.2
通讯作者：
Russo, C
中科院分区：
文献类型：
--
作者：
Nociari, MM;Shalev, A;Russo, C
在这项研究中，利用alamarBlue开发了一种荧光法，用于体外检测细胞介导的细胞毒性。AlamarBlue是一种无毒的活细胞代谢指标，在线粒体还原后变成荧光。通过比较含有效应细胞和靶细胞的井中活细胞的总数，以及分别播种靶细胞和效应细胞的井中活细胞的总数，来量化效应细胞对靶细胞的特异性裂解。同种异体反应性T细胞和自然杀伤细胞介导的细胞毒性活性已被检测到使用alamarBlue技术的新应用。我们开发的用于检测细胞介导的细胞毒性的检测方法非常敏感和特异性，并且比标准Cr-51检测方法需要的效应细胞数量要少得多。由于alamarBlue试剂对细胞无毒，且实验可以在无菌条件下进行，如果需要，可以在最后回收效应细胞进行进一步分析或细胞扩增。用alamarBlue法与标准Cr-51释放法测定细胞介导的细胞毒性，结果表明，前者的特异性和灵敏度均高于传统方法。此外，在alamarBlue细胞毒性测定中观察到非常小的测定间和测定内的差异。总之，本研究表明，alamarBlue测定是一种非常敏感、经济、简单和无毒的方法，用于评估细胞介导的细胞毒性，使用有限数量的效应细胞产生准确的结果。此外，由于该分析是一个一步的过程，并且不涉及人员的任何风险，它可能是有用的自动分析大量样品中细胞介导的细胞毒性。(C) 1998 Elsevier Science B.V.版权所有
In this study, a fluorometric method using alamarBlue has been developed for detecting cell-mediated cytotoxicity in vitro. AlamarBlue is a non-toxic metabolic indicator of viable cells that becomes fluorescent upon mitochondrial reduction. Specific lysis of targets by effector cells is quantified by comparing the total number of viable cells in wells containing effector and targets together, with wells where target and effector cells were separately seeded. Cell-mediated cytotoxic activity by alloreactive T cells and natural killer cells has been detected using a novel application of the alamarBlue technique. The assay that we have developed to detect cell-mediated cytotoxicity is extremely sensitive and specific and requires a significant lower number of effector cells than the standard Cr-51 assay. Since alamarBlue reagent is non-toxic to cells and the assay can be performed under sterile conditions, effector cells may be recovered at the end for further analysis or cell expansion, if desired. Direct comparison of cell-mediated cytotoxicity measured by the alamarBlue method with the standard Cr-51 release assay revealed that the former method is as specific and more sensitive than the conventional assay. Moreover, very small inter and intra-assay variations have been observed for alamarBlue cytotoxicity assays, In conclusion, this study shows that the alamarBlue assay is an extremely sensitive, economical, simple and non-toxic procedure to evaluate cell-mediated cytotoxicity that yields accurate results using a limited number of effector cells. Furthermore, since this assay is a one-step procedure, and does not involve any risk for the personnel, it may be useful to analyze automatically cell-mediated cytotoxicity in a large number of samples. (C) 1998 Elsevier Science B.V. All rights reserved.