Details
| Original language | English |
|---|---|
| Pages (from-to) | 441-9 |
| Number of pages | 9 |
| Journal | Journal of Bioenergetics and Biomembranes |
| Volume | 47 |
| Issue number | 5 |
| Publication status | Published - Oct 2015 |
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.
Keywords
- Animals, Carbenoxolone/pharmacology, Cattle, Cell Line, Endothelial Cells/cytology, Gap Junctions/metabolism, Gold/chemistry, Lasers, Metal Nanoparticles/chemistry, Rats
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In: Journal of Bioenergetics and Biomembranes, Vol. 47, No. 5, 10.2015, p. 441-9.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Gold nanoparticle-mediated (GNOME) laser perforation
T2 - a new method for a high-throughput analysis of gap junction intercellular coupling
AU - Begandt, Daniela
AU - Bader, Almke
AU - Antonopoulos, Georgios C
AU - Schomaker, Markus
AU - Kalies, Stefan
AU - Meyer, Heiko
AU - Ripken, Tammo
AU - Ngezahayo, Anaclet
PY - 2015/10
Y1 - 2015/10
N2 - 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.
AB - 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.
KW - Animals
KW - Carbenoxolone/pharmacology
KW - Cattle
KW - Cell Line
KW - Endothelial Cells/cytology
KW - Gap Junctions/metabolism
KW - Gold/chemistry
KW - Lasers
KW - Metal Nanoparticles/chemistry
KW - Rats
U2 - 10.1007/s10863-015-9623-y
DO - 10.1007/s10863-015-9623-y
M3 - Article
C2 - 26310434
VL - 47
SP - 441
EP - 449
JO - Journal of Bioenergetics and Biomembranes
JF - Journal of Bioenergetics and Biomembranes
SN - 0145-479X
IS - 5
ER -