Thank you Shafq!

Hi Denise,

As you already have learned that endothelial cell (EC) TEER is markedly lower (approx 10X) than that of most epithelial cells. ECs in culture are notorious for poor tightness/integrity of the monolayer, and represents a huge challenge for the researchers addressing EC physiology/pathophysiology employing in vitro models. Therefore it is quite challenging to get reproducible/reliable TEER readouts in EC cultures, particularly if one is using the classical "chopstick" electrodes for TEER measurements. I don't know what TEER measurement setup(s) you have in your lab, but we found that "Endohm" system (World precision Instruments) works much better that the "chopstick" electrodes from the same supplier. We also found that ECIS Cell-based Assays from Applied BioPhysics are way superior in addressing EC barrier integrity and allows to monitor EC monolayer integrity in real time without disturbing the cells. If, however, you do not have access to the latter system, I would suggest the following for reproducible measurements of your EC TEER : 1) if possible always use low passage number ECs (e.g. passage 1-5) , 2) avoid changing cell culture/experimental medium just before measuring TEER and minimize time of EC exposure to room air (if possible, never aspirate cell culture medium from the inserts completely during change of the medium!). Always allow cells to adapt for 10-15 min following change of the cell culture/experimental medium, 3) avoid using buffers without Ca2+/Mg2+ (e.g. PBS) for EC washing (if necessary for the experimental procedure) that quickly and markedly affect TEER, 4) cell culture medium/buffer temperature is critical for reliable TEER measurements. Even "room temperature" buffers will decrease EC TEER values!

Hope that will be be off help in your experiments.

Best of luck!

Gedas

Dear Gediminas,

I really appreciate your time and your insights are extremely helpful!

Thank you very much! I am going to try ECIS cell based and impedance. My TEER measurements are lower than 8, not reliable at all.

Thanks again!

Denise, my group is performing similar assays on endothelial barrier function using both HUVECs and CMECs and had similar issues attaining any reliable data using both tracer molecule methods (FITC-Dextran) and the portable EVOM unit that records TEER. We have recently gotten our hands on an ECIS-Z system and so far it has been fantastic. You can measure monolayer resistance in real-time and the entire system in non-invasive. The data has panned out quite nicely after the introduction of a TJ disruption agent such as TNF-a. If you can attain a system or purchase one I highly recommend one.