Hi, I trypsinize endothelial cell from glass coverslip and trypsinization took almost 30 minutes. However, the cell still attached on coverslip instead of floating. Any solution so that the cell easily trypsinize from glass coverslip?
Hello Yuenwah! According to my experience, it sounds strange to me that endothelial cells are still adherent after 30 min exposure to trypsin. Firstly, I suppose that you use trypsin/EDTA for the detachment. In addition, the solution should be stored in aliquots at -20oC so that you avoid continuous freeze-thawing process for trypsin/EDTA. Another important advice is that you should put your wells with coverslips in the incubator at 37oC because the whole process of detachment is becoming more quickly. 1-2 min in the incubator, then check your cells and follow this procedure again depending on the cell trypsination. I hope that I helped you! Good luck!
Kind Regards,
Vasileios
Hi Vasileios Chantzichristos. Yes, I did that, but after I detach, I still manage to observe the cell on glass coverslip.
I forgot something important! Another reason that may cause problem in your process is that the coverslips are glass and not plastic. Is there a great number of cells which are attached on your coverslip?
Dear Vasileios,
Yes, a great number of cells attached on the coverslip. However, I put 1mL of trypsin on the glass coverslip.
Dear Yuenwah Chan,
Do you wash your cells before trypsinize them? You could try 3 times washing with PBS (without calcium or magnesium). To wash away some of the cations (endothelial integrins use them to adhere to surfaces). The EDTA in the trypsin as suggested by Vasileios Chantzichristos does the same trick, but in our hands pre-washing 3 times with PBS works really good, Just add, swirl, remove, repeat. By washing you also remove serum remnants that inhibit trypsin. Never trypsinize them more than 10 minutes. The cells will start clumping together and you will damage them. After washing them with PBS, trypsinization with addtional EDTA for max 5 minutes at 37 degrees should be fine. And as an extra: a good (but gentle) smack with your bottle/plate againts the table or microscope will detach them completely. The cells can handle it. :)
Good luck.
GreetZ Michel
Thank you Michel W.J. Smeets for such a good explanation. After I wash it with PBS, the cell really easier to be detached.
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