Dear researchers,
We are trying to figure out a problem with our cells (caco2 and MDCK) and are hoping you can help us.
The permeability of our control compound Nadolol is 10 times higher than it was in the past. We tried a lot of different things, but the problem remains.
We tried thawing and seeding new cell lines from other frozen batches which showed good results in the past, we made new HBSS, we made new culture medium, we used different lots of serum, the pH of the media used is checked. The TEER value before the experiment is as expected, sometimes the TEER value drops after the experiment, but there are also experiments in which the TEER values after the experiment is the same as before the experiment. We also do a Na-fluorescein test at the end of the experiment. Based on that test, the permeability of the cells is to high. The amount of DMSO present in the samples added to the apical side of the cells is less than 0.1%.
At this moment we have not really an idea what to do next.
If any of you has any suggestions, please feel free to share with us.
Thanks!
Kind regards,
Sophie
I assume that your cells (caco2 and MDCK) are immortalized. They are expected to retain their endothelial properties, unlike primary cells. However, I have worked with immortalized bEnd.3 cells, and found that morphological changes were observed with extended passaging (more than passage 50). You can read similar findings in the following paper:
Article Tight junction protein expression and barrier properties of ...
Therefore, it is always a good idea to store the cells at every cell pasage in liquid nitrogen, especially those below passages 40.
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