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

Song Yang, Wen Hong Li

Research output: Contribution to journalArticle

10 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)94-101
Number of pages8
JournalNature Protocols
Volume4
Issue number1
DOIs
StatePublished - 2009

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Molecular Probes
Optical Imaging
Fluorescent Dyes
Cell Communication
Fluorescence
Chemical activation
Infrared radiation
Imaging techniques
Communication
Assays
Coloring Agents
Fluorophores
Photons
Connexins
Gap Junctions
Esters
Cells
Permeability

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

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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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