Trypsin detach adherent cells from dish so we can transfer them. When to stop trypsin by adding medium? Is seeing moving round cells enough? Should cells move fast or slow? Do cells must move single or is it ok to move in chains? I always see some cells in chain after trypsin for 5-8 min and I afraid to increase time not to affect cell vitality. Attached a diagram
thanks
It depends on what cells you are using. If you are using confluent epithelial cells such as MDCK, it takes more than 10 min or even as long as 30 min to completely detach the cells from dish, and 30 min trypsinisation does not kill the cells. If you could see almost all of the cells flowing in the trypsin solution, that's enough. In many cases several cells remain clustered. For daily culture that would not cause problem.
Hello Heba,
As Yutaka said, it depends entirely on your cell's type. Average time is 5-10 min, it could be as long as 20 min in some epithelial cells and as low as 2 min in some stem cells. You have to look into the literature and check the time used for your particular cell type.
As for the picture you provided, I believe that image c (chain of cells) is more realistic, since your cells in B might die from too much time in contact with trypsin (I wonder if these cells in image B grow after that much time?).
I know that we want to harvest every single cell on the plate, but this usually leads to bad results and cell death !!
alternately you could try double trypsinizations, meaning that you trypsinize for 5-10 min then add medium, collect floating cells and then do another trypsinization step. But this method will cost you more.
Best regards,
Ahmed
It depends on cell type. however, 5 mins is enough with gentle shake for most cases.
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