I usually use the cell dissociation buffer from Gibco, 15min, 37°C, but will switch to Versene.
Would you use Versene at 37°C during 15minutes?
Or even longer?
The presence of EDTA in trypsin aims at depleting extracellular calcium thus promoting dissociation in cells. However, long exposure leads to cell death often recognised by aberrant morphology. In my experience I have exposed Trypsin-EDTA for not more than 3 minutes (500ul in a small flask T25) for various epithelia cell lines (HeLa, Vero, fibroblast), MDCK and CaCo-2 for 5min. Tapping in between to assist lifting of cells as Glen mentioned should be applied as well. Another thing to consider, wash your cells with calcium and magnesium free PBS prior to trypsinisation as this enhances dissociation (i.e by removing Ca++ and Mg++ present in culturing media on cell surface). Inactivation of trypsin by 1:6 of trypsin: culturing media is optimal in my practise especially if you are planning to seed cells over 24 hours and treatment thereafter where the window period for recovery of cells is nil. Good luck!
thank you all for your answers!
I surely never use trypsin EDTA to detach cell before facs analysis, since it alters the cell surface markers.
I finally received my usual Gibco cell dissociation buffer. but it's good to know i can use Versene as well.
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