Flow cytometric analysis of fluorescein-labeled nerve growth factor binding to A875 human melanoma cells.
Flow cytometric analysis of fluorescein-labeled nerve growth factor binding to A875 human melanoma cells.
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流式细胞术分析荧光素标记的神经生长因子与 A875 人黑色素瘤细胞的结合。
DOI：
10.1006/excr.1994.1012
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发表时间：
1994
影响因子：
3.7
通讯作者：
Neet,KE
中科院分区：
文献类型：
--
作者：
Kasaian,MT;Jacobberger,JW;Neet,KE
荧光素标记的神经生长因子（NGF）与人黑色素瘤细胞（A875）的相互作用进行了研究，以评估更好的方法，严格的NGF结合研究。在与碳二亚胺和胱胺反应后，用碘乙酰胺荧光素在单个羧基上修饰NGF。与天然NGF相比，经修饰的NGF在与放射性标记的NGF的竞争中显示出完全的结合能力，并且在神经突生长测定中显示出完全的生物活性。使用流式细胞术测定与未固定的活细胞的结合。这种方法提供的优点是，未结合的配体不需要从细胞相关的配体中分离出来，从而避免了结合平衡的扰动，并且可以进行准确、广泛的统计分析。荧光素-NGF的结合主要是特异性的和可饱和的，通过三种数据处理方法的分析表明，在4°C下Kd为0.8至3 nMat。基于时间的数据采集允许产生连续的时间过程。结合在细胞暴露于配体的5分钟内达到稳态水平。用荧光素-NGF得到的动力学和稳态结果与以前的125 I-NGF结合研究的数据吻合得很好。NGF系统中流式细胞术方法的主要限制是与放射性标记的NGF结合相比相对缺乏灵敏度，部分原因是与NGF结合的荧光团的不寻常淬灭。
The interaction of fluorescein-labeled nerve growth factor (NGF) with human melanoma cells (A875) has been studied in order to assess better methodology for rigorous NGF binding studies. The NGF was modified at a single carboxyl group with iodoacetamidofluorescein after reaction with carbodiimide and cystamine. The modified NGF showed full binding competence in competition with radiolabeled NGF and full biological activity in neurite outgrowth assays compared to native NGF. Binding to unfixed, viable cells was assayed using flow cytometry. This method offers the advantage that unbound ligand need not be separated from that which is cell-associated, thus avoiding pertubation of the binding equilibrium, and accurate, extensive statistical analysis is possible. Binding of fluorescein-NGF was mainly specific and saturable, with analysis by three methods of data treatment indicating a Kdof 0.8 to 3 nMat 4°C. Time-based data acquisition allowed a continuous time course for binding to be generated. Binding reached a steady-state level within 5 min of exposure of the cells to the ligand. Kinetic and steady-state results obtained using fluorescein-NGF agree well with previous data produced by125I-NGF binding studies. The main limitation of the flow cytometric method in the NGF system is the relative lack of sensitivity compared to the binding of radiolabeled NGF, partially due to unusual quenching of the fluorophore bound to NGF.