TY - JOUR
T1 - Imaging of filter-grown epithelial cells
T2 - MDCK Na+-H+ exchanger is basolateral
AU - Rosenberg, S. O.
AU - Berkowitz, P. A.
AU - Li, L.
AU - Schuster, V. L.
PY - 1991
Y1 - 1991
N2 - We report a simple method for growing epithelial cells on permeable supports and for imaging the cells from the apical side using an inverted microscope. Madin-Darby canine kidney (MDCK) cells were either seeded onto the conventional side of Millipore-CM filters or onto ''inverted'' filters. The peak transepithelial resistance of confluent monolayers was the same with either system. Cells on inverted filters that were stained with various dyes and imaged by epifluorescence exhibited more distinct intercellular spaces, cell margins, nuclei, and subapical vesicles. We also perfused both sides of inverted filters with HCO3/CO2-free saline and measured intracellular pH (pH(i)) using 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) and digital imaging. The intrinsic buffer capacity of MDCK cells increased exponentially as pH(i) decreased. After an NH4Cl load, the H+ extrusion rate (J(H+)) in control saline was 2.42 ± 0.62 mM/min. J(H+) was completely blocked by 1 mM basolateral amiloride. In contrast, 1 mM apical amiloride had no effect. We conclude that 1) growth of epithelial cells on an inverted filter system is useful for the microspectrofluorimetric determination of pH(i) in single cells and for the imaging of apical/subapical structures, and 2) the Na+-H+ exchanger of MDCK cells is functionally polarized to the basolateral membrane.
AB - We report a simple method for growing epithelial cells on permeable supports and for imaging the cells from the apical side using an inverted microscope. Madin-Darby canine kidney (MDCK) cells were either seeded onto the conventional side of Millipore-CM filters or onto ''inverted'' filters. The peak transepithelial resistance of confluent monolayers was the same with either system. Cells on inverted filters that were stained with various dyes and imaged by epifluorescence exhibited more distinct intercellular spaces, cell margins, nuclei, and subapical vesicles. We also perfused both sides of inverted filters with HCO3/CO2-free saline and measured intracellular pH (pH(i)) using 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) and digital imaging. The intrinsic buffer capacity of MDCK cells increased exponentially as pH(i) decreased. After an NH4Cl load, the H+ extrusion rate (J(H+)) in control saline was 2.42 ± 0.62 mM/min. J(H+) was completely blocked by 1 mM basolateral amiloride. In contrast, 1 mM apical amiloride had no effect. We conclude that 1) growth of epithelial cells on an inverted filter system is useful for the microspectrofluorimetric determination of pH(i) in single cells and for the imaging of apical/subapical structures, and 2) the Na+-H+ exchanger of MDCK cells is functionally polarized to the basolateral membrane.
KW - 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein
KW - Digital imaging
KW - Epithelial transport
KW - Fluorescence microscopy
KW - Image analysis
KW - Intracellular pH
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U2 - 10.1152/ajpcell.1991.260.4.c868
DO - 10.1152/ajpcell.1991.260.4.c868
M3 - Article
C2 - 1850200
AN - SCOPUS:0025905189
SN - 0002-9513
VL - 260
SP - C868-C876
JO - American Journal of Physiology - Cell Physiology
JF - American Journal of Physiology - Cell Physiology
IS - 4 29-4
ER -