I am working with Caco-2 cells. I seed them on transwell filter. In our group TEER has been used to assess the the monolayer integrity. However TEER has been unreliable due to high flactuation. We do also BCA for total protein expression. if any one suggest a good method to varify the integrity?
It's always best to measure cell monolayer integrity using at least two methods if TEER is one of them. Typically one method involves flux of a solute that others have shown to be restricted by formation of the monolayer. How this is done depends upon what your lab can measure, e.g., a fluorescent or radiolabled solute. There are scores of publications on measuring permeability using Caco-2 cell monolayers and small- to large-sized solutes from Lucifer yellow to dextrans. Good luck!
Thank you Thomas.
We have used both metods: TEER and 4 kd FITC. However I am looking for more new and more reliable method.
These methods are quite reliable and, in my 20+ yrs experience with permeability measurements using epithelial and endothelial cell monolayers, what is typically unreliable are the cells themselves with Caco-2 being the most "temperamental". One can measure substantial TEER values before and after poking holes thru the filter with a dissecting needle whereas solute flux will increase exponentially. Hope you find what you're looking for.
Thank you Thomas, I agree Thomas. However, we had a lots of flactuation in TEER measurments. We are not using TEER.
We measure total protein by BCA assay.
I was also thinking of using zonulin experssion or ZO1 as a complementary. What do you think?
Any surrogate measure, e.g., total protein, etc, whether quantitative in itself or not, must still be correlated to a functional performance of tight junction formation, which is indicative of a polarized cell monolayer.
Thank you Thomas,
Thomas, I have another question:
can we use total protein for checking cell viability?
How accurate it would be?
Hi Frida,
Thanks for your answer . Yes I do. which proteins or genes can be targeted?
Radiolabelled mannitol has worked the best in our hands. Lucifer Yellow, FITC-Dextran 4kD, Inulin, PEG 5000 and others work. The key is to set thresh holds in poermeability that indicate the cells can be used. Dr. Raub was also correct about TEER. Temperature changes, buffer composition, chopstick placement, etc. can all influence it. I had a colleague once jokingly mix purified water with HBSS (50:50) to show his student that he could easily raise the TEER value with minimal effort. The student was showcked unteil the trick was revealed. Paracellular permeating solutes mimicking the size of the compounds you plan to study are best to normalize the cells.
As for viability: LDH, MTT, etc can all be used. Even simple Trypan blue cell staining can be used to adjust viable cell plating conditions. We have performed permeability studies with labeled paracellular permeants to show that our compound of interest does not alter cell integrity. Cell viability qRT-PCR arrays are also excellent.
Lastly, maks sure you check for mycoplasma on occasion. They are insideious and can dramatically effect results. These can be overlooked.
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