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

anyone know why there is EDTA in trypsin?

hi
EDTA has the same effect as trypsin , i mean we can detach cells using EDTA alone and its much gentler on cells than trypsin , so i think its added to trypsin to enhance its effect and i read also that it can decrease the clumbing of cells.
hope this help.

Actually trypsin/EDTA is a combined method for detaching cells. Trypsin cuts the adhesion proteins in cell-cell and cell-matrix interactions (i don't remember the specific site), and EDTA is a calcium chelator, which integrins needs to interact with other proteins for cell adhesion-- no calcium, no cell adhesion. And that's why EDTA treatment is gentler than trypsin.

Dear All,

Tissue culture media contains Calcium and Magnesium ions, foetal calf serum contains proteins that are trypsin inhibitors. Both Mg2+/Ca2+ INHIBIT TRYPSIN. The reason why we use PBS without Ca2+/Mg2+ to wash the cells prior to trypsinisation is to reduce the concentration of Divalent cations and proteins that inhibit trypsin action. EDTA is a Calcium chelator which will "mop" up the remaining divalent cations. If trypsin is allowed to stay in contact with the cells for too long a time, cell viabilty will reduce.
This should be the first principle of cell culture that you learn on day one. There are only very few cells that will detach with EDTA treatment alone.

for EDTA:
i remember when working with plant tissue culture, we used EDTA as a chelating agent in order to keep Mg and other elements in suspension so the plant tissue can easily absorb them.......

agree with rhombus ' explanation

Dear Spainishflower,

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.

yes, basics are usually ignored....

A bit off topic maybe (I agree completely with Rhombus on the fact function of EDTA and Trypsin), but most people nowadays aren't trained too much.

People just buy kits and buffer solutions without thinking what's in it, and what it all does. It's getting worse, because companies are making it "easier" by selling ready to use mixes (for instance for a PCR, you only have to add your primers and template and you're ready to go, it's automated hot start and all, so you hardly have to know what you're doing and what's in your tubes to do a succesfull PCR).
People hardly know how to work sterile, don't know how to measure pH correctly,... Makes it a lot harder if you need to troubleshoot any kind of reaction or process.

A PhD-student working here is working with DNA quite a lot, and she didn't even know what A260/280 ratio was and why it was important.

So, everybody: if you are teaching someone techniques (whatever they be), try to explain everything there is to know about the technique, no matter what "easy" technique it is for you (and experienced performer of it).

This is not meant to offend anybody, if you're not taught anything, you do the right thing by asking questions and I would urge everybody to try to find some answers by "googling" them, but if need be to come and ask them here.

I agree with both Rhombus and vairus that many students and technicians do not know the underlying theory behind the experiments.

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.