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A real-time method of imaging glucose uptake in single, living mammalian cells

Nature Protocols volume 2, pages 753–762 (2007)Cite this article

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Abstract

This protocol details a method for monitoring glucose uptake into single, living mammalian cells using a fluorescent D-glucose derivative, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), as a tracer. The specifically designed chamber and superfusion system for evaluating 2-NBDG uptake into cells in real time can be combined with other fluorescent methods such as Ca2+ imaging and the subsequent immunofluorescent classification of cells exhibiting divergent 2-NBDG uptake. The whole protocol, including immunocytochemistry, can be completed within 2 d (except for cell culture). The procedure for 2-NBDG synthesis is also presented.

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Figure 1: Analytical data of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG).
Figure 2: A culture dish–based chamber for live-cell imaging.
Figure 3
Figure 4: A custom-made holder for the outlet needle (Narishige).
Figure 5: Measurement of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) uptake into MIN6 cells.
Figure 6: Measurement of [Ca2+]i in response to glucose stimulation and subsequent 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) uptake in living pancreatic islet cells followed by immunocytochemical identification.

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Change history

  • 09 August 2007

    In the version of this article initially published, on p. 754 under “Reagents,” “N-(7-nitrobenz-2-oxa-1,3-diazol-4yl-)amino chloride” should have been “4-Chloro-7-nitrobenzofurazan”. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We are grateful to our collaborators, Drs. Masanori Nakata and Naoki Horimoto. We also thank Drs. K. Yoshizaki and S. Sato for technical help, and Dr. J. Miyazaki (Osaka University) for providing us with MIN6 cells.

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Authors and Affiliations

  1. Department of Physiology, Hirosaki University School of Medicine, Aomori, 036-8562

    Katsuya Yamada

  2. CREST of Japan Science and Technology Agency, Honcho 4-1-8, Kawaguchi, Saitama, 332-0012, Japan

    Katsuya Yamada

  3. Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo, 184-8588

    Mikako Saito & Hideaki Matsuoka

  4. CREST, Japan

    Mikako Saito & Hideaki Matsuoka

  5. Department of Physiology, Akita University School of Medicine, 1-1-1 Hondo, Akita, 010-8543

    Nobuya Inagaki

  6. Department of Diabetes and Clinical Nutrition, Kyoto University Graduate School of Medicine, Kyoto

    Nobuya Inagaki

  7. CREST, Japan

    Nobuya Inagaki

Authors
  1. Katsuya Yamada
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Correspondence to Hideaki Matsuoka or Nobuya Inagaki.

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Yamada, K., Saito, M., Matsuoka, H. et al. A real-time method of imaging glucose uptake in single, living mammalian cells. Nat Protoc 2, 753–762 (2007). https://doi.org/10.1038/nprot.2007.76

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