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Assaying dynamic cell–cell junctional communication using noninvasive and quantitative fluorescence imaging techniques: LAMP and infrared-LAMP

Nature Protocols volume 4, pages 94–101 (2009)Cite this article

Abstract

This protocol describes a fluorescence imaging assay, local activation of molecular fluorescent probes (LAMP), for measuring rates of intercellular dye transfer across gap junction channels in intact living cells. The LAMP method consists of four steps: (i) loading cells with a cell-permeable and photo-activatable fluorophore, NPE-HCCC2/AM (acetoxymethyl ester); (ii) locally photolyzing a caged coumarin in one cell of a coupled cell pair to release the parent fluorophore, HCCC2; (iii) imaging cell–cell transfer of HCCC2; and (iv) analyzing rates of dye diffusion. Compared with other methods available for measuring junctional coupling, the LAMP method offers a number of advantages, including noninvasiveness, ease of quantification of coupling strength, good temporal resolution and compatibility with multicolor imaging. Moreover, as the LAMP assay can be carried out multiple times in the same coupled cell pairs, changes in molecular permeability of connexin channels can be tracked by comparing rates of dye transfer. Finally, NPE-HCCC2 and HCCC2 have high two-photon uncaging and excitation efficiency, respectively, enabling two-photon uncaging and imaging to be combined to examine cell coupling in three dimensions (infrared-LAMP assay). It takes roughly 3 h or 4 h to complete a LAMP or an infrared-LAMP assay, respectively.

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Figure 1: Chemical and photochemical properties of a caged coumarin for live cell imaging.
Figure 2: Performing local uncaging using a field diaphragm.
Figure 3: LAMP assay of gap junctional communication.
Figure 4: Monitoring dynamic cell–cell dye transfer by two photon uncaging and imaging.

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Acknowledgements

This work is supported by grants from NIH and from the Welch foundation (I-1510).

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Author notes

  1. Song Yang

    Present address: Present address: Center for Research and Development of Fine Chemicals, Guizhou University, GuiYang 550025, People's Republic of China.,

Authors and Affiliations

  1. Department of Cell Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, 75390, Texas, USA

    Song Yang & Wen-Hong Li

  2. Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, 75390, Texas, USA

    Song Yang & Wen-Hong Li

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  1. Song Yang
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Correspondence to Wen-Hong Li.

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Supplementary Table

Supplementary Table 1: An example of processing cell-cell dye transfer data using Fick’s equation. (PDF 67 kb)

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Yang, S., Li, WH. Assaying dynamic cell–cell junctional communication using noninvasive and quantitative fluorescence imaging techniques: LAMP and infrared-LAMP. Nat Protoc 4, 94–101 (2009). https://doi.org/10.1038/nprot.2008.219

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