Purpose: 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.