I have been using Accutase as a trypsin like enzyme to dissociate adherent cells in a flask. To split the cells I first decant the media and rinse them with PBS, but after the rinse I added fresh PBS and Accutase together before I incubated them for 10 mins for optimum dissociation.
My cells grew fine and I've been doing it for several passages, but I was recently told that I should have never mixed Accutase with PBS. I checked the product info sheet for Accutase and I haven't seen anything regarding PBS. Has anyone done this before or has any info regarding PBS effects?
Any information would be appreciated.
Cheers,
Hi Thomas,
I was being conservative with the amount of Accutase I was using and their suggested amount barely covered my cells, so I added Accutase into PBS so my cells were completely covered. The total amount of Accutase exposed to the cells should be the same, regardless of the dillution.
I just wanted to make sure that there were non adverse effects, since I was told in a way that hinted as if the method was harmful to the cells.
Hi Yuka
sure, the total amount of the enzyme is not changed, but the concentration of the enzyme is changed due to dilution. Another proplem that you will face is the change of the pH of the original enzyme solution which will be changed also due to dilution by PBS. Also, you have to consider the concentration of enzyme per surface area.
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