A Novel Tool to Characterize Paracellular Transport: The APTS–Dextran Ladder
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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.
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.
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.
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.
Key wordsAPTS–dextran BBB blood–brain barrier cell layer tightness paracellular transport
astrocyte conditioned medium
brain microvascular endothelial cells
confocal laser scanning microscopy
- LIF detector
laser-induced fluorescence detector
transendothelial electrical resistance for studies with cell lines ECV304 and PBMEC/C1–2, transepithelial electrical resistance in case of Caco-2
von Willebrand factor