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

Nature Protocols
Song Yang, Wen-hong Li

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 a...Continue Reading

References

Feb 13, 2002·Quarterly Reviews of Biophysics·A L Harris
May 16, 2002·Journal of Cellular Physiology·Antonio De MaioJorge E Contreras
Nov 5, 2003·Nature Biotechnology·Warren R ZipfelWatt W Webb
Feb 26, 2004·Neuron·Michael V L Bennett, R Suzanne Zukin
Jun 1, 2005·Biochimica Et Biophysica Acta·Pauline Phelan
Apr 15, 2006·Cell Communication & Adhesion·Kenneth Dakin, Wen-Hong Li

❮ Previous
Next ❯

Citations

Oct 1, 2015·Toxicology in Vitro : an International Journal Published in Association with BIBRA·Michaël MaesMathieu Vinken
Jun 28, 2014·International Journal of Developmental Neuroscience : the Official Journal of the International Society for Developmental Neuroscience·Ibrahim AlsaeedAmer Kamal
Jul 29, 2009·Critical Reviews in Biochemistry and Molecular Biology·Mathieu VinkenVera Rogiers
Sep 7, 2016·Small·Yi-Feng WangXing-Jie Liang
Mar 12, 2010·Chemistry : a European Journal·Weiying LinLin Yuan
Mar 15, 2011·Current Protocols in Toxicology·Brad L Upham
Feb 15, 2021·Translational Research : the Journal of Laboratory and Clinical Medicine·Shlok JindalGopinath Packirisamy

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.