Why EDTA in trypsin? - (Oct/24/2006 )
Thank you for the woooow.
I have to say that this is very common place these days. Protocols are handed down and first principles are sometimes forgotten. I teach cell and tissue culture to all staff in my university department who want to learn from first principles. 9 times out of 10 the staff in question have had "training" of some description, but their ignorance is always apparent. They invariably come to me for help after having problems in their specific labs. For example :-
" I think their is a problem with my cells, they are not growing very fast "
" I think the tissue culture room is contaminated in some way, my cells are contaminated "
" I don't know what's wrong but my media is a funny colour "
All the above are easily handled but are regular events in most labs. I learned in the 1970's how to do cell culture on the bench with a bunsen burner, no HEPA filters and all re-usuable glass culture flasks and pipettes. It's just common sense.
I am fairly new to tissue culture and am working with HeLa cells, and understood that you are quite knowledgable.
My question is how many min does it need to detatch with trypsin-Edta and and why cells become less viable if they are kept too long ?
Wash the cells x2/x3 with PBS "A" (w/o Ca2+/Mg2+)
Then use trypsin..... normally 30 seconds-1 minute.
Some cell lines require longer. However always try other alternatives.....i.e. wash an additional x2 with EDTA prior to the PBS wash.
I was always taught that the trypsin, if left too long, would INTERNALISE and then degrade intracellular proteins...causing a loss in total viability.
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?
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?
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.
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
It is fine, if they get the expected results. However, when it come to troubleshooting, they do not know what to do and may expect their mentors/supervisors to solve it.
I am currently a second year PhD student, my PI expects results and don't care whether I understand the theory...he opposed to reading.
Not to criticise you directly, but your comment caught my attention. Please don't assume that all technicians have no interest in learning underlying theory and expect their supervisors to troubleshoot for them. I once worked for a Ph.D. who REFUSED to teach me to troubleshoot my own work. He told me that technicians are supposed to do the methods as directed, and that I didn't need to know the theory behind the method--that was his job.