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Title: Quantitative imaging of glutathione in live cells using a reversible reaction-based ratiometric fluorescent probe

item JIANG, XIQIAN - Baylor College Of Medicine
item YU, YONG - Baylor College Of Medicine
item CHEN, JIANWEI - Baylor College Of Medicine
item ZHAO, MINGKUN - Baylor College Of Medicine
item CHEN, HUI - Baylor College Of Medicine
item SONG, XIANZHOU - Baylor College Of Medicine
item MATZUK, ALEXANDER - Baylor College Of Medicine
item CARROLL, SHAINA - Baylor College Of Medicine
item TAN, XIAO - Baylor College Of Medicine
item SIZOVS, ANTONS - Baylor College Of Medicine
item CHENG, NIHGHUI - Children'S Nutrition Research Center (CNRC)
item WANG, MENG - Baylor College Of Medicine
item WANG, JIN - Baylor College Of Medicine

Submitted to: ACS Chemical Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/19/2014
Publication Date: 12/19/2014
Citation: Jiang, X., Yu, Y., Chen, J., Zhao, M., Chen, H., Song, X., Matzuk, A.J., Carroll, S.L., Tan, X., Sizovs, A., Cheng, N., Wang, M.C., Wang, J. 2014. Quantitative imaging of glutathione in live cells using a reversible reaction-based ratiometric fluorescent probe. ACS Chemical Biology. 10:864-874.

Interpretive Summary: Scientists always want to light up the molecules and see the changes inside the cells, which will greatly help to understand the biological process related to the normal growth or development of diseases. In the current study, we developed a tool to measure the rapid change of glutathione (GSH) in the cell, an important molecule to protect cells from oxidative damage. The scientists created a colorful chemical called CouBro which can stick to GSH after a quick chemical reaction, and then changed the color of the newly formed complex. By measuring the changes of the color in live cells, the scientists can determine whether the amount of GSH is increased or decreased following the changes of cell conditions. This colorful chemical can be widely used to monitor reduction and oxidation processes in different cells under nutritional interventions and provides a powerful tool for other scientists to study the mechanism of how cells manage pathways to control cellular metabolism.

Technical 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 understanding of cell biology and has become an indispensable tool in current biological studies. In order to minimize the perturbance on 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 reaction is completed. A reversible reaction-based probe with an appropriate equilibrium constant allows measurement of an analyte at much higher concentration, and thus, is a prerequisite for GSH quantification inside cells. In this contribution, we report the first fluorescent probe CouBro 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 CouBro concentrations as low as 50 nM. Furthermore, the GSH concentrations measured using CouBro in NIH-3T3, HeLa, HepG2, PANC-1, and PANC-28 cells are well correlated with the values obtained from cell lysates. CouBro imaging can also resolve the changes of GSH concentration in PANC-1 cells upon diethylmaleate (DEM) treatment. Through this study, we demonstrate the importance of reaction reversibility in designing quantitative reaction-based fluorescent probes.