2.4 Endothelial cell permeability tests
This experiment was completed with a Corning 24-well plate Transwell
chamber; 100μl of ECM was added into the upper chamber and then
incubated in a 37℃ cell incubator for 2h. After 2h, 100μl of cell
suspension (3.5 × 104 cells) was added to the upper
chamber and 600μl of ECM was added to the lower chamber. Once the
endothelial cells had formed a monolayer, we set up the groups described
above and cultured these four groups of cells with different stimulating
factors for 48 hours. Then, we discarded ECM in the upper and lower
chambers, added 200μl of FITC-dextran (1500μg/ml) to the upper chamber
and 600μl of fresh ECM to the lower chamber. After avoiding light and
incubating at 37℃ in a 5% CO2 incubator for 2h, we
extracted 10μl of sample from the lower chamber of each group and
transferred this to a black 96-well plate (10μl of sample solution +
90μl of fresh ECM). The fluorescence intensity of each sample was then
measured with a fluorescence spectrophotometer. The ratio of the
fluorescence intensity in each group to that of the control group was
used to represent the permeability of the endothelium.