A novel tool to characterize paracellular transport: the APTS-dextran ladder.
OBJECTIVE
The aim of this work was to develop an easy, manageable, and precise analytic tool to describe the tightness of cell layers by a molecular weight ladder.


METHODS
Dextrans were labeled by reductive amination with fluorescent 8-aminopyrene-1,3,6-trisulfonate (APTS). This mixture, including the internal standard diazepam, was used for transport studies in Transwell models using Caco-2, ECV304, and PBMEC/C1-2 cell lines. Samples were analyzed by fluorimetry, capillary electrophoresis, and reverse-phase high-performance liquid chromatography.


RESULTS
Following this approach, a logarithm correlation of R2 = 0.8958 between transepithelial electrical resistance (TEER) and APTS-dextran permeability was shown. In addition, a TEER-dependent permeability pattern could be observed including each single fraction from free APTS, APTS-glucose up to APTS-dextran consisting of 35 glucose units. The TEER-independent permeability coefficients of diazepam and confocal laser scanning microscopy images confirmed the paracellular transport of APTS-dextran.


CONCLUSIONS
All in all, the developed APTS-dextran ladder is a useful tool to characterize cell layer tightness and especially to describe paracellular transport ways and the extent of leakiness of cell layers (for blood-brain barrier or intestinal studies) over time--applying a wide array from smaller to larger molecules at the same time to refine TEER, sucrose, or Evans blue measurements.
Neuhaus
Winfried
W
Department of Medicinal Chemistry, University of Vienna, Pharmacy Center, Althanstrasse 14, A-1090, Vienna, Austria.
Bogner
Elisabeth
E
Wirth
Michael
M
Trzeciak
Joanna
J
Lachmann
Bodo
B
Gabor
Franz
F
Noe
Christian R
CR
D000818:Animals; D001812:Blood-Brain Barrier / Q000378:metabolism*; D018938:Caco-2 Cells; D002199:Capillary Permeability; D002463:Cell Membrane Permeability; D003911:Dextrans / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D003975:Diazepam / Q000378:metabolism; D004058:Diffusion; D017097:Electric Impedance; D019075:Electrophoresis, Capillary; D042783:Endothelial Cells / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis / Q000138:chemical synthesis / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D018613:Microscopy, Confocal; D008970:Molecular Weight; D011721:Pyrenes / Q000032:analysis / Q000138:chemical synthesis / Q000378:metabolism*; D013997:Time Factors
C513542:APTS-dextran; D003911:Dextrans; D005456:Fluorescent Dyes; D011721:Pyrenes; C015219:fluorescein isothiocyanate dextran; D016650:Fluorescein-5-isothiocyanate; D003975:Diazepam