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

Development of an optimized protocol for studying the interaction of filamentous bacteriophage with mammalian cells by fluorescence microscopy.
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DOI:
10.1002/jemt.20793
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发表时间:
2010-05
影响因子:
2.5
通讯作者:
Mao, Chuanbin
Mao, Chuanbin
中科院分区:
工程技术3区
文献类型:
--
作者:
Abbineni, Gopal;Safiejko-Mroczka, Barbara;Mao, Chuanbin

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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 (SEM).
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发表时间: 1996-03-28
期刊: NATURE
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通讯作者: Ruoslahti, E
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