Measurement of primary endothelial cell permeability to fluxes of dextran or albumin
The fluxes of albumin or dextran across vascular endothelial cell (ECs) were evaluated using Transwell chambers.
Both molecules were measured for the following reasons:
1) they have different sizes, with albumin having a molecular mass of ～65 kDa and dextran ～10 kDa;
2) they may use different transport mechanisms across ECs.
In addition to pinocytotic and intercellular transport, albumin transport can be modulated by albumin-binding proteins present on the EC surface.
ECs were seeded onto Transwell inserts (6.5-mm diameter and 0.4-μm pore size) coated with 12.5 μg/ml fibronectin.
One hour later, 0.05 μCi of 125I-labeled human albumin (Iso-Tex Diagnostics) and 250 μg of FITC-conjugated dextran (10 kDa) were added to the top well, and 100-μl aliquots of samples were taken from the bottom chamber every 15 min for 2 h.
The amounts of albumin and dextran in the samples were measured using a gamma counter and a fluorescence plate reader.
Each time after taking a sample, 100 μl of medium was added back to the bottom chamber.
For each time point, the amounts of albumin and dextran present in the bottom wells as well as the amounts in all the samples removed for measurement were calculated, added up, and plotted.
In every experiment, fibronectin-coated filter inserts without ECs were included, and the amounts of dextran and albumin in the bottom wells in each sample were expressed relative to the amounts of these two molecules in the filter-alone samples at 2 h.