title: Overlap of Doxycycline Fluorescence with that of the Redox-Sensitive Intracellular Reporter roGFP.
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.
authors.affiliation: Department of Pharmacology and the Center for Lung Biology, University of South Alabama School of Medicine, Mobile, AL, 36688, USA.
authors.forename: Heba
authors.initials: H
authors.lastname: Khader
authors.affiliation: Department of Pharmacology and the Center for Lung Biology, University of South Alabama School of Medicine, Mobile, AL, 36688, USA.
authors.forename: Victor
authors.initials: V
authors.lastname: Solodushko
authors.affiliation: Department of Pharmacology and the Center for Lung Biology, University of South Alabama School of Medicine, Mobile, AL, 36688, USA.
authors.forename: Abu Bakr
authors.initials: AB
authors.lastname: Al-Mehdi
authors.affiliation: Department of Microbiology and Immunology and the Center for Lung Biology, University of South Alabama School of Medicine, Mobile, AL, 36688, USA.
authors.forename: Jonathon
authors.initials: J
authors.lastname: Audia
authors.affiliation: Department of Pharmacology and the Center for Lung Biology, University of South Alabama School of Medicine, Mobile, AL, 36688, USA. bfouty@health.southalabama.edu.|Department of Internal Medicine and Division of Pulmonary and Critical Care Medicine, University of South Alabama, School of Medicine, Mobile, AL, 36688, USA. bfouty@health.southalabama.edu.|Center for Lung Biology, Department of Pharmacology, MSB 3406, University of South Alabama School of Medicine, Mobile, AL, 36688, USA. bfouty@health.southalabama.edu.
authors.forename: Brian
authors.initials: B
authors.lastname: Fouty
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004318:Doxycycline / Q000737:chemistry*; D005453:Fluorescence; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D010084:Oxidation-Reduction; D051381:Rats; D013050:Spectrometry, Fluorescence
chemical_list: D049452:Green Fluorescent Proteins; D004318:Doxycycline
keywords: Doxycycline; Fluorescent proteins; Fluorochromes; Tetracycline-inducible system; roGFP