I am using 0.05% Trypsin/EDTA. Added 2ml to T75 flask, incubated for 3min but not more than 10% cells were detached and there were very less circular cells. Not sure what was wrong.
You should wash the PBS twice and suggest that again wash with 1ml trypsin smoothly within 15 sec. and then add the trypsin for 2 min at 37 0C than observe till you are seeing the cells are coming up and then you add the media for stop the trysinization. It may take between 3-5 min. you should optimize it according to your conditions.
Washing with HBSS or PBS is necessary to remove the serum, we in our lab use 0.25% Trypsin/EDTA and typically need to leave 3ml of trypsin on the cells (T75) for 2-3 min and some time need to tap the flask gently. HUVECs detach very easily.
Hi,
Yes, definitely rinse with PBS or serum-free medium first. Then, 2-4 ml of trypsin, even at low concentration, should be sufficient to detach your HUVEC within 2-5 min at 37ºC. Firm but gentle tapping of the cell culture flask can also help to loosen them.
Also, I would avoid using EDTA-containing buffer to dilute the trypsin, but rather use PBS, as the EDTA is not required for detaching the cells and could actually stress them.
When you say "trypsinize for 3 min", do you guys start with warm trypsin (put in water bath)? Or start with cold trypsin?
Thanks!
During cell culture, PBS, Trypin and culture medium, all should be pre-warmed at 37 degree centigrade prior to use.
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