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Journal of Fluorescence

, Volume 24, Issue 2, pp 305–311 | Cite as

Overlap of Doxycycline Fluorescence with that of the Redox-Sensitive Intracellular Reporter roGFP

  • Heba Khader
  • Victor Solodushko
  • Abu Bakr Al-Mehdi
  • Jonathon Audia
  • Brian Fouty
SHORT COMMUNICATION

Abstract

Tetracycline-inducible systems allow for either suppression or induction of transgene expression to facilitate studies of cell physiology. Doxycycline is a preferred inducer for these gene expression systems due to its membrane permeability; however, the heterocyclic structure of doxycycline exhibits fluorogenic properties that can potentially bias measurement of other fluorochromes. Thus the simultaneous use of tetracycline-inducible systems and fluorescent proteins as reporter genes or as intracellular biosensors may lead to potentially confounding results. Herein, using cells which co-express the ratiometric redox sensitive intracellular reporter, roGFP, and a tetracycline-inducible reporter plasmid encoding the reporter gene, mCherry, as a model system, we describe the overlapping intracellular fluorescent signals between doxycycline and commonly used intracellular fluorescent probes. In our cells, the addition of doxycycline to cells caused a dose- and time-dependent increase in cell fluorescence with 405 nm excitation which overlapped with that of the oxidized configuration of roGFP. Incubating cells in concentrations of doxycycline less than 1 μg/mL and removing doxycycline from the media 60 min before performing experiments eliminated fluorescence interference while still maintaining maximal reporter transgene activation.

Keywords

Doxycycline roGFP Tetracycline-inducible system Fluorescent proteins Fluorochromes 

Notes

Acknowledgment

This work was supported, in part, by an American Heart Association Grant-in-Aid (AHA-271032) from the Southeastern region.

Disclosures/Conflict of interest statement

The authors declare no conflict of interest.

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Heba Khader
    • 1
  • Victor Solodushko
    • 1
  • Abu Bakr Al-Mehdi
    • 1
  • Jonathon Audia
    • 2
  • Brian Fouty
    • 1
    • 3
    • 4
  1. 1.Department of Pharmacology and the Center for Lung BiologyUniversity of South Alabama School of MedicineMobileUSA
  2. 2.Department of Microbiology and Immunology and the Center for Lung BiologyUniversity of South Alabama School of MedicineMobileUSA
  3. 3.Department of Internal Medicine and Division of Pulmonary and Critical Care MedicineUniversity of South Alabama, School of MedicineMobileUSA
  4. 4.Center for Lung Biology, Department of Pharmacology, MSB 3406University of South Alabama School of MedicineMobileUSA

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