I have infected MEFs for induction in vitro, but after culture over a week, I found the MEFs form a compact over the petri dish, the normal trypinisation doesn't work. I also tried collogenase IV, and then digest with trypsin, still doesn't work. Should I add some DNaseI in the digestion solution? Or any idea other methods for digestion? Or could any kind of matrix be added in the culture preventing the aggregate of the overgrown MEFs?
I would suggest that you increase your Trpysin/EDTA solution concentration and make sure that you have some DNase in there (because you will unfortunately have some cell lysis). In conjunction I would use a needle with syringe and draw the cell suspension up and down a few times to help break up the cell clusters. Finally passing the solution through a cell strainer to ensure you have dissociated cells for re-plating.
Normally the proliferation of MEFs should be self limiting so perhaps the infection has played some part in overriding this. You could probably passage before it become overconfluent in the future.
For particularly adherent cells which are recalcitrant to the standard protocol we use a fresh trypsin/EDTA solution buffered to pH8 with good results, since the pH enabling optimum activity of this enzyme is 7.8-8.7. pH8 is not dommageable for the cells for a short period of trypsinization. Hope this will be helpfull. Regards
If all else fails you could always use a cell scraper and try trypsinising the remaining cells.
I would suggest you to increase the trypsinisation time as well as concentration as mentioned by Sun Yung.
It is hard to believe that overgrown cultures are not getting trypisinized since these digesting agents you have used are also been used in tissue digestion. we often use to digest tissue which is more compact then monolayer overgrown cultures. although there are few steps to be taken care to ensure you are doing it right.
1) wash the cells thoroughly to remove serum contents or components which hinder the activity of digesting enzyme
2) digestion is possible when the temperature is at 37C so ensure that the enzyme must be warm before addition and after addition must be kept at 37C for few minutes till you see the digestion
3) Use the right percentage of digestion for stubborn cells like fibroblasts. This must help.
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