Time-resolved fluorometric assay for natural killer activity using target cells labelled with a fluorescence enhancing ligand
Time-resolved fluorometric assay for natural killer activity using target cells labelled with a fluorescence enhancing ligand
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DOI：
10.1016/0022-1759(96)00063-4
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发表时间：
1996-06-21
影响因子：
2.2
通讯作者：
Akerman, K
中科院分区：
文献类型：
--
作者：
Blomberg, K;Hautala, R;Akerman, K
描述了用荧光增强配体2,2':6‘，2n -三吡啶-6,6’ -二羧酸（TDA）的乙酰氧基甲酯标记的靶细胞来测量自然杀伤细胞活性的时间分辨荧光测定法。TDA的疏水酯化形式（双（乙酰氧基甲基）2,2':6',2 " -三吡啶-6,6 " -二羧酸酯，BATDA）很容易扩散通过活细胞的细胞膜。BATDA被细胞内酯酶水解，导致靶细胞内不透膜的TDA积累。标记的K-562细胞与效应细胞孵育后，从裂解细胞中释放的TDA与Eu3+螯合到上清中。然后通过测量形成的EuTDA螯合物的强烈荧光来量化自然杀伤细胞的活性。与BATDA孵育时，靶细胞被快速标记，TDA从靶细胞中释放的速度比Cr-51快，自发释放允许建立短期释放试验，EuTDA的检测速度快（5分钟/96孔板）。此外，这种非放射性方法允许使用复杂的培养基，因为与基于提示荧光法的方法相比，使用时间分辨荧光法可以避免自体荧光的问题。
A time-resolved fluorometric assay for the measurement of natural killer cell activity against target cells labelled with the acetoxymethyl ester of the fluorescence enhancing ligand 2,2':6',2N-terpyridine-6,6''-dicarboxylic acid (TDA) is described. The hydrophobic esterified form of TDA (bis(acetoxymethyl)2,2':6',2 ''-terpyridine-6,6 ''-dicarboxylate, BATDA) diffuses readily through the cell membrane of viable cells. BATDA is hydrolysed by intracellular esterases resulting in accumulation of membrane impermeable TDA inside the target cells. After incubation of labelled K-562 cells with effector cells the TDA released from lysed cells into the supernatant is chelated with Eu3+. The natural killer cell activity is then quantified by measuring the intense fluorescence of the EuTDA chelates formed. Target cells are rapidly labelled when incubated with BATDA, TDA is released from target cells faster than Cr-51, the spontaneous release permits a short-term release assay to be set up and the detection of EuTDA is fast (5 min/96 well plate). Furthermore, this non-radioactive method permits the use of complex culture media since, in contrast to methods based on prompt fluorometry, the problem with autofluorescence can be avoided by the use of time-resolved fluorometry.