Gold nanoparticle-mediated (GNOME) laser perforation: a new method for a high-throughput analysis of gap junction intercellular coupling
- 310 Downloads
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.
KeywordsScrape loading Dye transfer Gap junction Gold nanoparticle-mediated laser perforation GNOME In vivo Vascular Endothelial cells
The authors thank Dr. Sabrina Schlie-Wolter for the kind gift of the RBE4 cells. The authors also thank Kristina Schmitt and Anne Klett for their assistance with the experiments.
- Carbone SE, Wattchow DA, Spencer NJ, Hibberd TJ, Brookes SJ (2014) Damage from dissection is associated with reduced neuro-musclar transmission and gap junction coupling between circular muscle cells of guinea pig ileum, in vitro. Front Physiol 5:319. doi: 10.3389/fphys.2014.00319 CrossRefGoogle Scholar
- Schomaker M, Fehlauer H, Bintig W, Ngezahayo A, Nolte, I, Murua-Escobar, H, Lubatschowski H, Heisterkamp A (2010) Fs- laser cell perforation using gold nanoparticles of different shapes. Proceedings of SPIE 7589:75890C-75890C-5Google Scholar
- Schomaker M, Killian D, Willenbrock S, Heinemann D, Kalies S, Ngezahayo A, Nolte I, Ripken T, Junghanss C, Meyer H, Escobar HM, Heisterkamp A (2014) Biophysical effects in off-resonant gold nanoparticle mediated (GNOME) laser transfection of cell lines, primary- and stem cells using fs laser pulses. J Biophotonics 9999. doi: 10.1002/jbio.201400065
- Werkmeister E, Kerdjoudj H, Marchal L, Stoltz JF, Dumas D (2007) Multiphoton microscopy for blood vessel imaging: new non-invasive tools (Spectral, SHG, FLIM). Clin Hemorheol Microcirc 37(1–2):77–88Google Scholar