This protocol is a robust technique to determine the functional integrity of intercellular tight junctions in a cell model of the human blood-brain barrier. This is a simple and fairly inexpensive protocol that allows calculating the apparent permeability in a shorter time period instead of running a longer kinetic assay. This method can also be used to determine the functional integrity of tight junctions in brain endothelial cells transfected with polymer DNA nanoparticles.
To begin cell plating, place tissue culture inserts with microporous membranes into a 24-well culture plate. Add 400 microliters of 0.15-milligrams-per-milliliter collagen type one in each tissue culture insert. Then incubate for one hour in a 37 degree Celsius, 5%carbon dioxide incubator.
Rock the 24-well plate gently to allow even spreading of the collagen solution over the microporous membrane in the tissue culture inserts. After an hour, remove the collagen solution and gently wash the microporous membrane with 0.4 milliliters of 1X PBS buffer. Now, plate hCMEC/D3 cells with a density of 50, 000 cells per square centimeter in the cell inserts.
Place the 24-well plate with tissue culture setup in the incubator to allow cell attachment and proliferation. Incubate the plate for seven days to allow the cells to reach 100%confluency. Remove the growth medium every other day and transfer 0.5 milliliters of pre-warmed fresh media into the tissue culture inserts.
Repeat the plating procedure for Western blotting to determine changes in ZO-1 expression for DNA nanoparticle transfection, and for the ATP assay to determine cell viability in transfected cells. For determining Lucifer Yellow apparent permeability, on each day post-seeding, remove the growth medium and add 1.5 milliliters of pre-warmed transport buffer to the basolateral side of the well. Now add 58.3 microliters of 20-micromolar Lucifer Yellow solution to the apical side of each trans-well insert.
Save 50 microliters of the solution for fluorescence measurements. After completely removing residual PBS buffer from the apical side, add Lucifer Yellow solution as quickly as possible to avoid drying the cells. Ensure accurate volumes of Lucifer Yellow solution in the apical side.
Make sure to add the same and accurate volume of Lucifer Yellow solution in all inserts. Work quickly to avoid drying the cells. Incubate the cells in a rotary plate shaker at 100 RPM and 37 degrees Celsius for 60 minutes.
Then remove 30 microliters of the Lucifer Yellow sample from each apical compartment. Transfer the 20-micromolar Lucifer Yellow solution and the apical side samples to pre-labeled tubes. Now dilute the sample tenfold using transport buffer.
Remove 500 microliters from each basolateral compartment, and transfer the sample to pre-labeled tubes. Prepare a series of Lucifer Yellow standards for the standard curve. Add 100 microliters of each standard apical and basolateral sample to each well of a black 96-well plate in duplicate.
Use a fluorescence microplate reader to measure the Lucifer Yellow fluorescence intensity and calculate the apparent permeability, as described in the manuscript text. The effect of culturing time on Lucifer Yellow permeability was used to determine the apparent kinetics of tight junction formation. The apparent permeability values significantly decreased on day seven compared with day one, suggesting that the barrier became tighter.
The apparent permeability values then stabilized until day 10. This implied the barrier formation was complete and functional, resulting in decreased Lucifer Yellow paracellular transport. Western blotting was used to detect changes in the expression of the tight junction protein ZO-1 over time.
The two bands represent the two ZO-1 isoforms. Densitometry analysis revealed that the pixel value of ZO-1 increased from days three through seven post-seeding, suggesting that the tight junction protein ZO-1 formed continually from days three through seven. After calcium depletion treatment on day seven post-seeding, the band of ZO-1 was almost undetectable, indicating that ZO-1 was unable to form in the absence of calcium ions.
As shown in the results, Western blotting of ZO-1 protein can be performed to determine changes in the expression level of tight junction proteins, complementing the Lucifer Yellow-based functional activity data. We also discovered that the functional activity of tight junctions in human brain endothelial cells was not affected by DNA nanoparticle transfection.
