Overlap of Doxycycline Fluorescence with that of the Redox-Sensitive Intracellular Reporter roGFP
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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.
KeywordsDoxycycline roGFP Tetracycline-inducible system Fluorescent proteins Fluorochromes
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.
- 7.Produit-Zengaffinen N, Davis-Lameloise N, Perreten H, Becard D, Gjinovci A, Keller PA, Wollheim CB, Herrera P, Muzzin P, Assimacopoulos-Jeannet F (2007) Increasing uncoupling protein-2 in pancreatic beta cells does not alter glucose-induced insulin secretion but decreases production of reactive oxygen species. Diabetologia 50(1):84–93PubMedCrossRefGoogle Scholar
- 11.Jahnen-Dechent W, Ketteler M (2012) Magnesium basics. CKJ: Clin Kidney J 5(suppl 1):i3–i14Google Scholar
- 17.Franklin TJ (1966) Mode of action of the tetracyclines. Biochemical studies of antimicrobial drugs. Sixteenth Symposium of the Society for General Microbiology. Cambridge University Press, CambridgeGoogle Scholar