title: Quantitative imaging of glutathione in live cells using a reversible reaction-based ratiometric fluorescent probe.
abstract: Glutathione (GSH) plays an important role in maintaining redox homeostasis inside cells. Currently, there are no methods available to quantitatively assess the GSH concentration in live cells. Live cell fluorescence imaging revolutionized the field of cell biology and has become an indispensable tool in current biological studies. In order to minimize the disturbance to the biological system in live cell imaging, the probe concentration needs to be significantly lower than the analyte concentration. Because of this, any irreversible reaction-based GSH probe can only provide qualitative results within a short reaction time and will exhibit maximum response regardless of the GSH concentration if the reaction is completed. A reversible reaction-based probe with an appropriate equilibrium constant allows measurement of an analyte at much higher concentrations and, thus, is a prerequisite for GSH quantification inside cells. In this contribution, we report the first fluorescent probe-ThiolQuant Green (TQ Green)-for quantitative imaging of GSH in live cells. Due to the reversible nature of the reaction between the probe and GSH, we are able to quantify mM concentrations of GSH with TQ Green concentrations as low as 20 nM. Furthermore, the GSH concentrations measured using TQ Green in 3T3-L1, HeLa, HepG2, PANC-1, and PANC-28 cells are reproducible and well correlated with the values obtained from cell lysates. TQ Green imaging can also resolve the changes in GSH concentration in PANC-1 cells upon diethylmaleate (DEM) treatment. In addition, TQ Green can be conveniently applied in fluorescence activated cell sorting (FACS) to measure GSH level changes. Through this study, we not only demonstrate the importance of reaction reversibility in designing quantitative reaction-based fluorescent probes but also provide a practical tool to facilitate redox biology studies.
authors.affiliation: †Department of Pharmacology, ‡Department of Molecular and Human Genetics and Huffington Center on Aging, §Integrative Molecular and Biomedical Sciences Graduate Program, ∥USDA/ARS Children Nutrition Research Center and Department of Pediatrics, ⊥Center for Drug Discovery, Dan L. Duncan Cancer Center, and Cardiovascular Research Institute, Baylor College of Medicine, Houston, Texas 77030, United States.
authors.forename: Xiqian
authors.initials: X
authors.lastname: Jiang
authors.affiliation: †Department of Pharmacology, ‡Department of Molecular and Human Genetics and Huffington Center on Aging, §Integrative Molecular and Biomedical Sciences Graduate Program, ∥USDA/ARS Children Nutrition Research Center and Department of Pediatrics, ⊥Center for Drug Discovery, Dan L. Duncan Cancer Center, and Cardiovascular Research Institute, Baylor College of Medicine, Houston, Texas 77030, United States.
authors.forename: Yong
authors.initials: Y
authors.lastname: Yu
authors.affiliation: †Department of Pharmacology, ‡Department of Molecular and Human Genetics and Huffington Center on Aging, §Integrative Molecular and Biomedical Sciences Graduate Program, ∥USDA/ARS Children Nutrition Research Center and Department of Pediatrics, ⊥Center for Drug Discovery, Dan L. Duncan Cancer Center, and Cardiovascular Research Institute, Baylor College of Medicine, Houston, Texas 77030, United States.
authors.forename: Jianwei
authors.initials: J
authors.lastname: Chen
authors.affiliation: †Department of Pharmacology, ‡Department of Molecular and Human Genetics and Huffington Center on Aging, §Integrative Molecular and Biomedical Sciences Graduate Program, ∥USDA/ARS Children Nutrition Research Center and Department of Pediatrics, ⊥Center for Drug Discovery, Dan L. Duncan Cancer Center, and Cardiovascular Research Institute, Baylor College of Medicine, Houston, Texas 77030, United States.
authors.forename: Mingkun
authors.initials: M
authors.lastname: Zhao
authors.affiliation: †Department of Pharmacology, ‡Department of Molecular and Human Genetics and Huffington Center on Aging, §Integrative Molecular and Biomedical Sciences Graduate Program, ∥USDA/ARS Children Nutrition Research Center and Department of Pediatrics, ⊥Center for Drug Discovery, Dan L. Duncan Cancer Center, and Cardiovascular Research Institute, Baylor College of Medicine, Houston, Texas 77030, United States.
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
authors.affiliation: †Department of Pharmacology, ‡Department of Molecular and Human Genetics and Huffington Center on Aging, §Integrative Molecular and Biomedical Sciences Graduate Program, ∥USDA/ARS Children Nutrition Research Center and Department of Pediatrics, ⊥Center for Drug Discovery, Dan L. Duncan Cancer Center, and Cardiovascular Research Institute, Baylor College of Medicine, Houston, Texas 77030, United States.
authors.forename: Xianzhou
authors.initials: X
authors.lastname: Song
authors.affiliation: †Department of Pharmacology, ‡Department of Molecular and Human Genetics and Huffington Center on Aging, §Integrative Molecular and Biomedical Sciences Graduate Program, ∥USDA/ARS Children Nutrition Research Center and Department of Pediatrics, ⊥Center for Drug Discovery, Dan L. Duncan Cancer Center, and Cardiovascular Research Institute, Baylor College of Medicine, Houston, Texas 77030, United States.
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Matzuk
authors.affiliation: †Department of Pharmacology, ‡Department of Molecular and Human Genetics and Huffington Center on Aging, §Integrative Molecular and Biomedical Sciences Graduate Program, ∥USDA/ARS Children Nutrition Research Center and Department of Pediatrics, ⊥Center for Drug Discovery, Dan L. Duncan Cancer Center, and Cardiovascular Research Institute, Baylor College of Medicine, Houston, Texas 77030, United States.
authors.forename: Shaina L
authors.initials: SL
authors.lastname: Carroll
authors.affiliation: †Department of Pharmacology, ‡Department of Molecular and Human Genetics and Huffington Center on Aging, §Integrative Molecular and Biomedical Sciences Graduate Program, ∥USDA/ARS Children Nutrition Research Center and Department of Pediatrics, ⊥Center for Drug Discovery, Dan L. Duncan Cancer Center, and Cardiovascular Research Institute, Baylor College of Medicine, Houston, Texas 77030, United States.
authors.forename: Xiao
authors.initials: X
authors.lastname: Tan
authors.affiliation: †Department of Pharmacology, ‡Department of Molecular and Human Genetics and Huffington Center on Aging, §Integrative Molecular and Biomedical Sciences Graduate Program, ∥USDA/ARS Children Nutrition Research Center and Department of Pediatrics, ⊥Center for Drug Discovery, Dan L. Duncan Cancer Center, and Cardiovascular Research Institute, Baylor College of Medicine, Houston, Texas 77030, United States.
authors.forename: Antons
authors.initials: A
authors.lastname: Sizovs
authors.affiliation: †Department of Pharmacology, ‡Department of Molecular and Human Genetics and Huffington Center on Aging, §Integrative Molecular and Biomedical Sciences Graduate Program, ∥USDA/ARS Children Nutrition Research Center and Department of Pediatrics, ⊥Center for Drug Discovery, Dan L. Duncan Cancer Center, and Cardiovascular Research Institute, Baylor College of Medicine, Houston, Texas 77030, United States.
authors.forename: Ninghui
authors.initials: N
authors.lastname: Cheng
authors.affiliation: †Department of Pharmacology, ‡Department of Molecular and Human Genetics and Huffington Center on Aging, §Integrative Molecular and Biomedical Sciences Graduate Program, ∥USDA/ARS Children Nutrition Research Center and Department of Pediatrics, ⊥Center for Drug Discovery, Dan L. Duncan Cancer Center, and Cardiovascular Research Institute, Baylor College of Medicine, Houston, Texas 77030, United States.
authors.forename: Meng C
authors.initials: MC
authors.lastname: Wang
authors.affiliation: †Department of Pharmacology, ‡Department of Molecular and Human Genetics and Huffington Center on Aging, §Integrative Molecular and Biomedical Sciences Graduate Program, ∥USDA/ARS Children Nutrition Research Center and Department of Pediatrics, ⊥Center for Drug Discovery, Dan L. Duncan Cancer Center, and Cardiovascular Research Institute, Baylor College of Medicine, Houston, Texas 77030, United States.
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
mesh_terms: D041721:3T3-L1 Cells; D000818:Animals; D004847:Epithelial Cells / Q000378:metabolism* / Q000648:ultrastructure; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D005978:Glutathione / Q000032:analysis* / Q000378:metabolism; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D010084:Oxidation-Reduction
chemical_list: D005456:Fluorescent Dyes; D005978:Glutathione