TY - JOUR
T1 - Assaying dynamic cell-cell junctional communication using noninvasive and quantitative fluorescence imaging techniques
T2 - LAMP and infrared-LAMP
AU - Yang, Song
AU - Li, Wen Hong
N1 - Copyright:
Copyright 2009 Elsevier B.V., All rights reserved.
PY - 2009
Y1 - 2009
N2 - 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.
AB - 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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U2 - 10.1038/nprot.2008.219
DO - 10.1038/nprot.2008.219
M3 - Article
C2 - 19131961
AN - SCOPUS:61449098545
SN - 1754-2189
VL - 4
SP - 94
EP - 101
JO - Nature Protocols
JF - Nature Protocols
IS - 1
ER -