best method for detaching cells - in culture (May/30/2005 )
What is the best method to detach adherent cells in culture?
... incubating them with trypsin/edta @ 37 degrees, 5 min? then flapping flask against bench...
.... incubating them with pbs/edta @ 37 degrees for 30 min?
i use tyrpsin-EDTA for 4-5 mins at room temp. If the cells are really sticky and not coming off ...then 37C fro 5 mins. This is for routine cell culture..It depends on the experiments..for eg for westerns I scrape the cells
I avoid tapping the flask...
It depends on your cells, some cells will no tolerate rough treatment, such as trypsin treatment, though short exposures won't hurt generally. I incubate cells with trypsin either at room temperature or at 37 until they start to detach with a bit of tapping. This usally occurs within 5 minutes. Never go over 10 minutes with trypsin as it does horrible things to the cells (expression, morphology, you name it) if you repeatedly do it.
Tapping can cause clumps of cells to detach, which can be difficult to separate into single cells, but pipetting up and down a few times will usually solve this. Another cause of clumping is over-confluent cell layers, the only solution is to avoid this.
just regarding your time procedures, i would prefer Trypsin.... Caus i think 30' in PBS may dammage cells. In my lab we always do a trypsinisation for passaging cells, diluted up to 1/5 in EDTA and incubate 37° / 5'
thanks for your feedback.
I also think that trypsin has been standarized for these purposes. But the thing is that cells that I am trying to culture (blood monocytes) seem to be too adhered to plastic and does not detach after 5 min trypsin @ 37 degrees, nor tapping, nor other 5 min... etc.
I have read a few protocols suggesting pbs/edta, that's why I wrote...
Again, many thanks!!