title: Gold nanoparticle-mediated (GNOME) laser perforation: a new method for a high-throughput analysis of gap junction intercellular coupling.
abstract: The present report evaluates the advantages of using the gold nanoparticle-mediated laser perforation (GNOME LP) technique as a computer-controlled cell optoperforation to introduce Lucifer yellow (LY) into cells in order to analyze the gap junction coupling in cell monolayers. To permeabilize GM-7373 endothelial cells grown in a 24 multiwell plate with GNOME LP, a laser beam of 88 μm in diameter was applied in the presence of gold nanoparticles and LY. After 10 min to allow dye uptake and diffusion through gap junctions, we observed a LY-positive cell band of 179 ± 8 μm width. The presence of the gap junction channel blocker carbenoxolone during the optoperforation reduced the LY-positive band to 95 ± 6 μm. Additionally, a forskolin-related enhancement of gap junction coupling, recently found using the scrape loading technique, was also observed using GNOME LP. Further, an automatic cell imaging and a subsequent semi-automatic quantification of the images using a java-based ImageJ-plugin were performed in a high-throughput sequence. Moreover, the GNOME LP was used on cells such as RBE4 rat brain endothelial cells, which cannot be mechanically scraped as well as on three-dimensionally cultivated cells, opening the possibility to implement the GNOME LP technique for analysis of gap junction coupling in tissues. We conclude that the GNOME LP technique allows a high-throughput automated analysis of gap junction coupling in cells. Moreover this non-invasive technique could be used on monolayers that do not support mechanical scraping as well as on cells in tissue allowing an in vivo/ex vivo analysis of gap junction coupling.
authors: Institute of Biophysics, Leibniz University Hannover, Herrenhäuser Str. 2, D-30419, Hannover, Germany.
authors: Daniela
authors: D
authors: Begandt
authors: Institute of Biophysics, Leibniz University Hannover, Herrenhäuser Str. 2, D-30419, Hannover, Germany.
authors: Almke
authors: A
authors: Bader
authors: Biomedical Optics Department, Laser Zentrum Hannover e. V., Hannover, Germany.
authors: Georgios C
authors: GC
authors: Antonopoulos
authors: Biomedical Optics Department, Laser Zentrum Hannover e. V., Hannover, Germany.
authors: Markus
authors: M
authors: Schomaker
authors: Biomedical Optics Department, Laser Zentrum Hannover e. V., Hannover, Germany.
authors: Stefan
authors: S
authors: Kalies
authors: Biomedical Optics Department, Laser Zentrum Hannover e. V., Hannover, Germany.|Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany.
authors: Heiko
authors: H
authors: Meyer
authors: Biomedical Optics Department, Laser Zentrum Hannover e. V., Hannover, Germany.
authors: Tammo
authors: T
authors: Ripken
authors: Institute of Biophysics, Leibniz University Hannover, Herrenhäuser Str. 2, D-30419, Hannover, Germany. ngezahayo@biophysik.uni-hannover.de.|Center for Systems Neuroscience Hannover, University of Veterinary Medicine Hannover Foundation, Hannover, Germany. ngezahayo@biophysik.uni-hannover.de.
authors: Anaclet
authors: A
authors: Ngezahayo
mesh_terms: D000818:Animals; D002229:Carbenoxolone / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism*; D006046:Gold / Q000737:chemistry*; D007834:Lasers; D053768:Metal Nanoparticles / Q000737:chemistry*; D051381:Rats
chemical_list: D006046:Gold; D002229:Carbenoxolone
keywords: Dye transfer; Endothelial cells; GNOME; Gap junction; Gold nanoparticle-mediated laser perforation; In vivo; Scrape loading; Vascular