Development of an optimized protocol for studying the interaction of filamentous bacteriophage with mammalian cells by fluorescence microscopy.
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.
Abbineni
Gopal
G
Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, Oklahoma 73019, USA.
Safiejko-Mroczka
Barbara
B
Mao
Chuanbin
C
D002460:Cell Line; D004847:Epithelial Cells / Q000648:ultrastructure*; D006801:Humans; D017912:Inovirus / Q000648:ultrastructure*; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D013194:Staining and Labeling / Q000379:methods*; D053585:Virus Attachment