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Flow Cytometry Protocols pp 355–370Cite as

Noncytotoxic DsRed Derivatives for Whole-Cell Labeling

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 699))

Abstract

Fluorescent proteins (FPs) are invaluable tools for biomedical research. Useful FPs have desirable fluorescence properties such as brightness and photostability, but a limitation is that many orange, red, and far-red FPs are cytotoxic when expressed in the cytosol. This cytotoxicity stems from aggregation. To reduce aggregation, we engineered the surface of DsRed-Express to generate DsRed-Express2, a highly soluble tetrameric FP that is noncytotoxic in bacterial and mammalian cells. Directed evolution of DsRed-Express2 yielded the color variants E2-Orange, E2-Red/Green, and E2-Crimson. These variants can be used to label whole cells for single- and multi-color experiments employing microscopy or flow cytometry. Methods are described for reducing the higher-order aggregation of oligomeric FPs and for analyzing FP cytotoxicity in Escherichia coli and HeLa cells.

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Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Gordon Center for Integrated Sciences, The University of Chicago, Chicago, IL, USA

    Rita L. Strack & Robert J. Keenan

  2. Department of Molecular Genetics and Cell Biology, Cummings Life Science Center, The University of Chicago, Chicago, IL, USA

    Benjamin S. Glick

Authors
  1. Rita L. Strack
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  2. Robert J. Keenan
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  3. Benjamin S. Glick
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Corresponding author

Correspondence to Benjamin S. Glick .

Editor information

Editors and Affiliations

  1. Medical Center, Flow Cytometry Core Facility, George Washington University, I Street NW 2300, Washington, 20037, District of Columbia, USA

    Teresa S. Hawley

  2. Medical Center, Dept. Anatomy & Regenerative Biology, George Washington University, I Street NW 2300, Washington, 20037, District of Columbia, USA

    Robert G. Hawley

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Strack, R.L., Keenan, R.J., Glick, B.S. (2011). Noncytotoxic DsRed Derivatives for Whole-Cell Labeling. In: Hawley, T., Hawley, R. (eds) Flow Cytometry Protocols. Methods in Molecular Biology, vol 699. Humana Press. https://doi.org/10.1007/978-1-61737-950-5_17

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  • DOI: https://doi.org/10.1007/978-1-61737-950-5_17

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61737-949-9

  • Online ISBN: 978-1-61737-950-5

  • eBook Packages: Springer Protocols

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