Hi all,
I am facing a problem to trypsinize and combine/move the transfected cells into new well . I was planning to pool the transfected cells into a new well to ensure better cell-cell contact, in hoping the cells can proliferate better. But every time after I trypsinize them, the transfected cells would die afterwards.
I have also included untransfected (normal) cells during the trypsinization and the normal cells do not die and proliferate well after trypsinization and well transferring.
Any way to solve this issue?
What is the concentration of trypsin. Try to use more diluted trypsin concentration and allow longer time for trypsin to work
Hi, the trypsin I used was aliquoted from the purchased stock and I didn't dilute it. I used the same trypsin to subculture cells, trypsinize untransfected cells etc. and those cells did not die but only transfected cells died..
People have used different concentration of cells to trypsinize cells. If cells are grown in serum free medium, diluted concentration is needed. You can try few diluted concentration of trypsin. Read on your bottle what is the concentration of trypsin.
Try to dilute trypsin in Hank's Balanced Salt Solution (Product No H9394, Sigma) or
buy diluted trypsin and try.
Try diluted and cold trypsin (4C), it will take more time but may not kill cells.
Hi,
It is absolutely normal for transfected cells to die. Transfected cells are already stressed further transferring then to new plates makes them more vulnerable.
We would transfect undifferentiated cells. These were then transfer to the new plate followed by differentiation once they have adhered to the plate.
So, u can keep a few things in mind for your experiment:
1. Transfect a relatively large number of cells, keeping in mind that some will die. ( i.e. do not transfer the exact number of cells as required for the new plate)
2. If your experiment involves differentiation of cells, transfect the undifferentiated cells, post transferring them to new plate initiate differentiation.
3. When tyrpsinising, be very gentle with the cells, as mentioned by Subhash use cold trypsin and try to finish the process of transferring cells in less than the usual time taken.
All the best !
Hi Suraiya Saleem & Subhash C. Juneja , thanks for the comments ..
For the cold trypsin, our practice here is to thaw the trypsin at room temperature and during trypsinization, we will incubate the cells inside the CO2 incubator at 37 degree celsius.. Thus, for your suggestion, should I trpysinize the cells inside 4 degree freezer before I centrifuge the cells?
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