I'm growing 5637 cells for the first time. During splitting, I noticed that after 2 min in 1x trypsin-EDTA, the cells are still adhering to the bottom of the flask. Do I need to use a higher concentration of the enzyme or increase the reaction time?
I think you can use slow hand taping of TC Flask after 2-3 min incubation of Trypsin EDTA with monlayer.
Did you rinse once with Trypsin-EDTA (or at least with PBS) to remove serum or proteins in the medium? You may try longer trypsinization, but should check under the microscope. Usually one should observe some changes in morphology, beginning losening or rounding up of adherent cells, then you may use slow or fast hand taping of the culture flask, or rinse with the pipette (and media) to remove remaining adherent cells.
Thank you Dr Shah and Dr Eibl. I usually rinse the cells with 1x HBSS before adding trypsin-EDTA. I'll try longer trypsinization this time.
I incubated the cells with 1x trypsin-EDTA for 10 minutes at 37C. They were still adherent.
You could try the following: strong tapping the flask against your other hand (not breaking the flask). Sometimes it may help to put the cells on ice for a minute, or use a cell scraper.
You may also check your trypsin - it is useful to aliquot the original flask and avoid repeated thawing and freezing, since the trypsin may digest itself with too long at room temperature.
Hope this helps.
Thank you so much for your help! After trying many times, I found that incubating the cells with 1x trypsin-EDTA for 15-18 minutes at 37C gives a good result.
If you are using trypsin-EDTA, make sure to rinse the cells before with PBS that does not contain any Ca or Mg ions because it is a chelating agent.
Also check the concentration of trypsin that you are using and incubate at 37C.
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