International Journal of Stem Cells

  • 제12권2호
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  • Pages.367-379
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  • 2019
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  • 2005-3606(pISSN)
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  • 2005-5447(eISSN)
한국줄기세포학회 (Korean Society for Stem Cell Research)
권호 표지
DOI QR코드

DOI QR Code

Monitoring Glutathione Dynamics and Heterogeneity in Living Stem Cells

  • Jeong, Eui Man (Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine) ;
  • Shin, Ji-Woong (Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine) ;
  • Lim, Jisun (Department of Biomedical Sciences, University of Ulsan College of Medicine) ;
  • Kim, Ju Hwan (Department of Chemistry, Korea University) ;
  • Kang, Hyewon (Metabolab. Inc.) ;
  • Yin, Yingfu (Cell2in, Inc.) ;
  • Kim, Hye-Mi (Cell2in, Inc.) ;
  • Kim, YongHwan (Cell2in, Inc.) ;
  • Kim, Sun-Gi (Cell2in, Inc.) ;
  • Kang, Heun-Soo (Cell2in, Inc.) ;
  • Shin, Dong-Myung (Department of Biomedical Sciences, University of Ulsan College of Medicine) ;
  • Choi, Kihang (Department of Chemistry, Korea University) ;
  • Kim, In-Gyu (Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine)
  • 투고 : 2018.12.28
  • 심사 : 2019.01.20
  • 발행 : 2019.07.31
⟨ 이전 논문 다음 논문 ⟩

초록

Glutathione (GSH) is a major antioxidant in cells, and plays vital roles in the cellular defense against oxidants and in the regulation of redox signals. In a previous report, we demonstrated that stem cell function is critically affected by heterogeneity and dynamic changes in cellular GSH concentration. Here, we present a detailed protocol for the monitoring of GSH concentration in living stem cells using FreSHtracer, a real-time GSH probe. We describe the steps involved in monitoring GSH concentration in single living stem cells using confocal microscopy and flow cytometry. These methods are simple, rapid, and quantitative, and able to demonstrate intracellular GSH concentration changes in real time. We also describe the application of FreSHtracer to the sorting of stem cells according to their GSH content using flow cytometry. Typically, microscopic or flow cytometric analyses of FreSHtracer and MitoFreSHtracer signals in living stem cells take ~2~3 h, and the fractionation of stem cells into subpopulations on the basis of cellular GSH levels takes 3~4.5 h. This method could be applied to almost every kind of mammalian cell with minor modifications to the protocol described here.

키워드

과제정보

We thank the staff of the Biomedical Imaging Center and FACS Center of the Core Research Facilities of Seoul National University College of Medicine for their technical advice and assistance. This work was funded by the National Research Foundation of Korea through the Basic Science Research Program (NRF-2017R1D1A1B03035059, 2017R1D1A1B03031379, NRF-2017M3A9B4061890, and NRF-2018R1A2B3008541); the Korea Healthcare Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (HI14C3339 and HI18C2396); and the Brain Korea 21 PLUS program of the Korean Ministry of Education, Science and Technology.

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