APTS-labeled dextran ladder: a novel tool to characterize cell layer tightness.
The aim of this work was the development of an easy manageable analytic system for describing tightness of cell layers in a molecular size dependent manner, which is more precise than currently used ones. Dextrans were labeled by reductive amination with fluorescent 1-aminopyrene-3,6,8-trisulfonate (APTS). This mixture, including internal standard diazepam, was used for transport studies, which were accomplished with an established transwell blood-brain barrier model culturing an immortalized porcine brain microvascular endothelial cell line (PBMEC/C1-2). Samples were analyzed by fluorescence measurements, capillary electrophoresis and RP-LC. Following this approach, a permeability pattern could be achieved including each single fraction from APTS, APTS-glucose to APTS-dextran consisting of 31 glucose units. Permeability coefficients were calculated and ranged from 16.38+/-3.79 microm/min for APTS to 6.07+/-1.23 microm/min for the APTS-dextran with 31 glucose units (diazepam: 67.97+/-7.32 microm/min). All in all, the developed APTS-dextran ladder is an useful tool to characterize cell layer tightness--especially to describe paracellular transport ways and leakiness status of the blood-brain barrier over time--applying a wide range from smaller to larger molecules at the same time in order to refine, e.g. TEER, sucrose or Evans blue measurements.
Neuhaus
Winfried
W
Department of Medicinal/Pharmaceutical Chemistry, University of Vienna, Pharmacy Center, Althanstrasse 14, A-1090 Vienna, Austria.
Trzeciak
Joanna
J
Lauer
Regina
R
Lachmann
Bodo
B
Noe
Christian R
CR
D000818:Animals; D001812:Blood-Brain Barrier / Q000378:metabolism*; D002199:Capillary Permeability; D002460:Cell Line; D003911:Dextrans; D017097:Electric Impedance; D019075:Electrophoresis, Capillary; D042783:Endothelial Cells / Q000378:metabolism*; D005456:Fluorescent Dyes; D011721:Pyrenes; D013552:Swine; D019108:Tight Junctions
C513542:APTS-dextran; D003911:Dextrans; D005456:Fluorescent Dyes; D011721:Pyrenes