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 μm/min for APTS to 6.07 ± 1.23 μm/min for the APTS-dextran with 31 glucose units (diazepam: 67.97 ± 7.32 μm/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.