Endothelial permeability - looking for help with my project - (Feb/15/2012 )

Hi there,

I have started my PhD project one year ago in the field of matrix biology. It still doesn't really have a research question, however, I am supposed to work on endothelial permeability.
I wanted to have an in vitro system to test the effect of a substance X we have purified in the lab on the permeability of endothelial cell monolayers. Unfortunately, the test (which is done in membrane inserts for 24 well plates and uses a fluorescently labeled dextran as tracer) didn't work. Which in this case means that I couldn't increase the permeability with any substance I tested (e.g. histamine, VEGF, EDTA, bradykinin). So after one year I still don't have any results and I need to think about a change in the project. My supervisor is not really helpful, he only suggests further reading (which I have done but which doesn't really help me having ideas). I already started working with mice and looking at the effect of substance X on tumor vascularure.

I really need some help/input here, because my funding ends on the 30th April and I will only get more money if I have results and a project plan for the next year. At the moment I am thinking of just giving up because I am so frustrated...

Any suggestions are most welcome!

Cheers,
hot_chocolate

Attached is an interesting paper related to monolayer permeability. They used texas red-labeled dextran but likely had a different set up. You can also measure TEER. All the info and methods is in the link below.

http://www.ncbi.nlm.nih.gov/pubmed?term=Transcellular%20Passage%20of%20Neisseria%20meningitidis%20across%20a%20Polarized%20Respiratory%20Epithelium

Good luck!

Look up trypan blue permeability assay or how you could use permeability chambers for performing endothelial cell permeability assay using albumin texas red. Hope this helps!