I want to test the effect on endothelial permeability of certain compounds. I am using Transwell filters (0.4 microns pore size), FITC conjugated dextran (40 kDa) and Human Aortic Endothelial Cells (HAEC) to try to perform a macromolecular permeability assay. Since I don't know if the compounds I intend to test will have an effect or not, I attempted to use TNF-alpha as a positive control to evaluate the protocol's effectiveness. I chose TNF-alpha because It came up frequently when searching for molecules capable of inducing endothelial permeability. The thing is I fail to spot any difference between treated and untreated cells. I don't know how long should I allow cells to grow on the Transwell before beginning the treatment.
I have attempted this twice. The first time, I allowed cells to grow for a single before beginning treatment (24 hrs of TNF-alpha at 10 ng/mL) and then added the fluorescent dextran to the top compartment to see how much was able to pour through in each condition, but saw no significant differences. The second time, I allowed them to grow for 3 days before beginning treatment, increased TNF-alpha concentration to 100 ng/mL and added a deprivation step (0.5 % FBS in comparison to the 10% I used the first time). I failed to see any difference this time either.
I'm attaching a .pptx with the details of what I did in case it helps. Some people doing this with Caco-2 cells wait for 21 days after seeding on the Transwell before performing the treatment. I don't know if it could be the same with these cells.
Also, in case it is important, on the day of treatment I swap media by using a vacuum to remove the old and just pipette from the top the rest. Is this protocol very delicate? Transwells don't offer very good visibility of cells so I don't know exactly what is going on with cells after I have seeded them. Perhaps if you aren't extremely delicate when doing this cells may detach or get sucked up by the vacuum. Does anyone know if this is the case?
I don't think the time is the important factor. I would think that you'd want a very confluent endothelial cell monolayer. The time it takes to achieve this will depend on the density at which you seed the cells.
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