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Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial enzyme which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG's to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20-30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes.
Pubmed ID: 27935478 RIS Download
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View all literature mentionsCell line HeLa is a Cancer cell line with a species of origin Homo sapiens
View all literature mentionsCell line CHO-K1 is a Spontaneously immortalized cell line with a species of origin Cricetulus griseus (Chinese hamster)
View all literature mentionsCell line U2OS is a Cancer cell line with a species of origin Homo sapiens (Human)
View all literature mentionsThis unknown targets GFP
View all literature mentionsThis unknown targets PMP70
View all literature mentionsThis monoclonal targets eCFP, CFP, mCerulean,eGFP, wtGFP, GFP S65T, AcGFP, TagGFP, tagGFP2, sfGFP, pHluorineYFP, YFP, Venus, Citrine
View all literature mentionsThis unknown targets
View all literature mentionsMulti paradigm numerical computing environment and fourth generation programming language developed by MathWorks. Allows matrix manipulations, plotting of functions and data, implementation of algorithms, creation of user interfaces, and interfacing with programs written in other languages, including C, C++, Java, Fortran and Python. Used to explore and visualize ideas and collaborate across disciplines including signal and image processing, communications, control systems, and computational finance.
View all literature mentionsThis unknown targets GFP
View all literature mentionsThis monoclonal targets eCFP, CFP, mCerulean,eGFP, wtGFP, GFP S65T, AcGFP, TagGFP, tagGFP2, sfGFP, pHluorineYFP, YFP, Venus, Citrine
View all literature mentionsThis unknown targets PMP70
View all literature mentionsThis unknown targets
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