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Development of an optimized protocol for studying the interaction of filamentous bacteriophage with mammalian cells by fluorescence microscopy

机译：通过荧光显微镜研究用于研究丝状噬菌体与哺乳动物细胞相互作用的优化方案的开发

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Filamentous bacteriophage has been proposed as a vehicle that can carry and deliver therapeutics into mammalian cells for disease treatment, thus a protocol for imaging phage-cell interaction is essential. Because high signal intensity is necessary to study the mechanism of interaction between filamentous bacteriophage and mammalian cells, it is important to optimize the procedure for fluorescence labeling of phage in order to understand such interaction. Here, we describe a procedure that gives intense fluorescence labeling and can show interactions between fd-tet bacteriophage selected from phage libraries and mammalian cells (SKBR-3 and MCF-10A). The indirect labeling of phage with dye-conjugated antibody and cytoskeleton associated proteins was significantly enhanced in the presence of a cross-linking reagent called dithiobissuccinimidylpropionate (DSP) as shown by qualitative and quantitative fluorescence microscopy. The use of DSP resulted in high signal intensity in fluorescence imaging of phage-cell complex. The DSP cross-linker is believed to preserve soluble unbound proteins for fluorescence imaging. The interaction between the phage and mammalian cells was further confirmed by scanning electron microscopy. Microsc. Res. Tech. 73:548-554, 2010.
机译：已提出丝状噬菌体作为可以将治疗剂携带和递送到哺乳动物细胞中以进行疾病治疗的载体，因此用于成像噬菌体-细胞相互作用的方案是必不可少的。因为高信号强度对于研究丝状噬菌体和哺乳动物细胞之间的相互作用机制是必需的，所以优化噬菌体的荧光标记程序以理解这种相互作用非常重要。在这里，我们描述了一种程序，该程序可以进行强烈的荧光标记，并且可以显示从噬菌体库中选出的fd-tet噬菌体与哺乳动物细胞（SKBR-3和MCF-10A）之间的相互作用。定性和定量荧光显微镜显示，在存在称为二硫代双琥珀酰亚胺基丙酸丙酸酯（DSP）的交联剂的情况下，通过染料偶联抗体和细胞骨架相关蛋白对噬菌体的间接标记显着增强。 DSP的使用在噬菌体-细胞复合物的荧光成像中产生了高信号强度。据信DSP交联剂可保存可溶性未结合的蛋白质用于荧光成像。噬菌体与哺乳动物细胞之间的相互作用通过扫描电子显微镜进一步证实。 Microsc。 Res。科技73：548-554，2010。

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