I am currently working with endothelial and epithelial cells and I need to test different Transwell materials (Polycarbonate, Teflon, Polyethylene terephthalate) for a diabetic model. All three materials are used in literature for similar models, however, I would like to test if, among them, there is one better than the other.
My goal is to generate a monolayer of both endothelial and epithelial cells on Transwell to obtain the most physio-pathological model as possible.
I was thinking about metabolic and proliferation assays to evaluate their "happiness". Thus I could perform some IF to stain tight junctions.
Does anyone have any suggestions?
Thank you so much.
Gaia
I think you may want to start by performing a TEER measurement to check that the tight junctions between the cells have formed.
other than that MTT assay is pretty standard for accessing cell proliferation so that might be a good place to start. Hope this helps!
One obvious, maybe too obvious, is simple microsopy. At least this is what I did evaluating different coating procedures... I used to fix cells with 4% PFA and stained cells with phalloidin to get an idea of what the actin cytoskeleton looked like. "Blebbing", for instance, gave a good indication of whether cells felt happy. Judgement of focal adhesions and stress fibres is another way of judging of how the cells do - apart from simple judgement of time to confluency. Hope this helps.
I think yours are good choices on the happiness side. I would think about unhappiness assays in addition. Metabolism and proliferation may not be enough, when it goes together with lots of death. Annexin stain for apoptosis. 7AAD for death. Detachment. Time of trypsinization required for detachment, to assess strength of adhesion (keep temperature constant!).