I have endothelial cells which I trypsinize routinely. But after treatment with some ER stressors like thapsigargin, cells adhere so tightly that it is almost impossible for me to detach in 10 minutes with 0.25% trypsin. They take much longer times. What could be the reason?
Ammar,
Thapsigargin increases intracellular Ca2+ concentrations in endothelial cells, so the question is how this will affect the cell adhesion?
The increase of the intracellular Ca2+ concentration will activate integrin-mediated adhesion leading to a tight adhesion of these cells to culture surface.
Maybe you need to apply a dose and time dependent manner of (Thapsigargin treatment with different incubation time) on these cells and check for the downstream applications, cell adhesion assays and expression of adhesion proteins.
- We used to induce ERS using tunicamycin not Thapsigargin, but again this will depend upon your research question and the downstream effects that you are investigating.
- I will not recommend using the cells for further experiments if kept with Trypsin-EDTA for too long, as you know this will damage the cell membrane and definitely, they will die! When collecting the cells, we need to wash it with fresh media or PBS to remove any remaining Trypsin-EDTA. Otherwise, you can collect the cells by scraping instead of using Trypsin-EDTA.
*Read also about:
The effect of Thapsigargin in releasing Inorganic phosphate (Pi) and the attached Review: Cell Cycle Regulation by Integrin-Mediated Adhesion.
They might interest you.
Good luck,
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