Roden M M, Lee K H, Panelli M C, Marincola F M
Surgery Branch, National Cancer Institute, Bethesda, MD 20892, USA.
J Immunol Methods. 1999 Jun 24;226(1-2):29-41. doi: 10.1016/s0022-1759(99)00039-3.
A novel cellular cytotoxicity assay using Calcein acetoxymethyl (Calcein-AM), a cytoplasmic fluorescent label, has been developed as an alternative to the standard 51Chromium (Cr)-release. Target cells were loaded with Calcein-AM and then co-incubated with effector cells. An additional reagent, FluoroQuench, is added to extinguish fluorescence of dying target cells and of the culture media. Assay plates are read on a quantitative fluorescent scanner for determination of viable target cells. Percent lysis is calculated as one minus the percent viable cells as compared to fluorescent-labeled targets-only wells. The assay was tested to demonstrate the lytic activity of cytotoxic T lymphocyte (CTL) cultures, lymphokine-activated killer (LAK), and natural killer (NK) cell line effectors against peptide-pulsed and melanoma targets. In addition to the acquisition of results comparable to the 51Cr-release assay, the Calcein assay reliably measures cell-mediated cytotoxicity with little variance among replicates. The fluorescent assay represents a simple and useful alternative to the use of radioactive materials and adds the additional benefit of digital images and analysis.
一种使用细胞质荧光标记物乙酰氧基甲基钙黄绿素（Calcein-AM）的新型细胞毒性测定方法已被开发出来，作为标准的51铬（Cr）释放法的替代方法。将靶细胞用Calcein-AM加载，然后与效应细胞共同孵育。添加另一种试剂FluoroQuench以淬灭垂死靶细胞和培养基的荧光。在定量荧光扫描仪上读取测定板以确定存活的靶细胞。裂解百分比的计算方法是，与仅含荧光标记靶细胞的孔相比，用1减去存活细胞百分比。该测定方法经过测试，以证明细胞毒性T淋巴细胞（CTL）培养物、淋巴因子激活的杀伤细胞（LAK）和自然杀伤（NK）细胞系效应物对肽脉冲和黑色素瘤靶标的裂解活性。除了获得与51Cr释放测定法相当的结果外，Calcein测定法还能可靠地测量细胞介导的细胞毒性，各重复之间的差异很小。荧光测定法是使用放射性材料的一种简单而有用的替代方法，并且还具有数字图像和分析的额外优势。
