Why EDTA in trypsin? - (Oct/24/2006 )

As mentioned earlier, only a few cells are detqched using EDTA only.

I'm tryinging to trypsinise a cell line to get single cell suspension for FACS. I've tried teh following to no avail:

1) EDTA only (5mM)= clumped cells which cannot be counted evenafter vigorous pipetting
2) Enzyme-Free Cell Dissociation Solution = clumped cells, same as above

I will try using trypsin alone. I did not attempt this first as my previous cell line formed clumps with trypsin and dissociated well using EDTA only. Why are some cell lines morew difficult to dissociate then others at the same confluency? MY ESCC cell ine takes a very longtime dissociating with trypin and hence the reason I was avoiding using this for cell dissociation for FACS and long exposure trypsin may change affet cell viabiity and surface characteristics.

Just thought I'd mention

I work with Adherent cells all the time... Human, mouse, rat... endothelial, epithelial, etc...

I almost never use trypsin.

I simply use EDTA in buffer.. (PBS, HBSS, HBS, TRIS-HCL) at a concentration of 3 to 5 milli molar... I have never had trouble getting cells to detach...

Trypsin is faster (effective in about a minute or two) whereas using EDTA can take 5 to 10 minutes for cells to completely detach...

The advantage of using EDTA without Trypsin is that EDTA does NOT cleave protein. I study cell surface receptors and protein. If I were to use Trypsin I'd risk cleaving many of the protein I'm trying to study. With EDTA, the cells simply detach, then I spin and resuspend the cells in buffer without EDTA and i'm ready to proceed.

Unless you REALLY need cells to detach very quickly, trypsin (in my opinion) is an unecessary expense... But I'm constantly reminded that I do not know everything...

Hello cell culture enthusiasts,
Anyone knows a way to prevent cell aggregation while dissociating them with trypsin and alike?
Thnx

If you're centrifuging and removing the trypsin after you stop the reaction, you'll end up with a cell pellet. Make sure that is thoroughly broken up by repeated mixing with the micropipettor you use to resuspend the pellet. Also, you can try repeatedly washing the inside of the flask with the media you add to deactivate the trypsin, which should also break up clumps.

I was thinking more about preventative means to dissociate single cells in nice dispersed form without having to mechanically break the clumps by pipetting. I assume there are some anti-clumping additives for the dissociation solutions?

Hi Rhombus,

Do you know any books or websites, that present the basic information about the cell cullture techniques and problem solving.

Because what you said is true, we are concentrating on perform the techniqe with out knowing why.

I really would like to put my hand on some usefull books on tissue and cell culture.

Any recommendations??????????

Thanks

EDTA removes Ca and Mg ions from the medium. this metal ions inhibit trypsin. moreover, Ca is important for cell attachment to the substrate, so removing it cells easly detache. I usually wash cells with PBS without Ca/Mg to remove these ions and FBS (wich inhibits trypsin as well!!!) and then I add trypsin. cells detach very easly