Employment of confocal microscopy for the dynamic visualization of domes in intact epithelial cell cultures.
Many epithelial cells cultured on plastic ware form domes, fluid-filled localized raisings of the cell monolayer. Domes are due to active vectorial ion transport and their presence demonstrates the maintenance of a differentiated polarized phenotype and of tight junctional complexes. Through a confocal laser microscope equipped with a special flow chamber, intact domes were evaluated in real time for prolonged experimental periods. Both in CAPAN-1 pancreatic duct adenocarcinoma cells and in renal tubular LLC-PK1 cells, vertical sections of calcein-loaded cultures provided a clear visualization of dome outlines during the slow deflation induced by specific agonists (respectively, 1 microM secretin or 10 microM vasopressin). Section series of calcein-loaded domes were used for three-dimensional reconstructions. In CAPAN-1 cultures, cell depolarization induced by secretin was detected with the potentiometric dye bis-oxonol. In the same cells pyranine, a fluid phase marker that is cell impermeant, visualized dome compartment and paracellular pathways, also providing an evaluation of dome fluid pH. Confocal laser scanning microscopy of domes represents a convenient device for the functional assessment of living epithelial cells.
Rotoli
B M
BM
Department of Experimental Medicine, Section of General and Clinical Pathology, University of Parma, Parma, Italy.
Orlandini
G
G
Gatti
R
R
Dall'Asta
V
V
Gazzola
G C
GC
Bussolati
O
O
D001190:Arylsulfonates / Q000737:chemistry; D002460:Cell Line; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology* / Q000502:physiology; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008564:Membrane Potentials; D018613:Microscopy, Confocal / Q000379:methods*
D001190:Arylsulfonates; D005452:Fluoresceins; D005456:Fluorescent Dyes; C005047:pyranine; C007740:fluorexon