Why EDTA in trypsin? - (Oct/24/2006 )
you are totally right vairus - those who are not interested in what they are really doing - for instance what the role of each specific buffer component is - will be caught at the end if they should reflect on their data, discuss about their findings or write an exam;
but there is not only to blame lazy or untalented PhD students; often Prof have lost that for what they have got their position; horrible for those who are supervised by a Prof who has lost any contact to every day laboratory work and give "good" advices to solve f.i. method problems
Hi All,
Cadherins are a class of proteins which are expressed on the surface of cells. They play important roles in cell adhesion whereby they ensure cells within tissues or in monolayer are bound together. They are dependent on calcium (Ca2+) ions to function. By chelating Ca2+, cadherins can be inactivated and that helps in cell or tissue dissocoation.
Do you agree guys!!
you all are right. I think we should know how to do experiments,but more importantly,know why to do this!
trypsin is a serine protease...calcium ions do not inhibition trypsin....
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.
Yeah, Ca2+ should not inhibit trypsin. As mentioned, the Ca2+ is ESSENTIAL for integrin mediated cell attachment to the substratum. Integrins contain multiple solvent exposed Ca2+ binding sites. For more info on integrin structure, including metal binding see here http://www.sciencemag.org/cgi/content/full/294/5541/339#R17
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 come back to the very first question... does anyone know what percentage is better for the cells? trypsine-edta 0.25% or 0.05%?
Does that depend on the cell strength to stick to the plate?
Or is 0.25% a stock solution that I have to dilute before using directly on the cells? If so, what do I dilute it in?
Lol thanks if someone can answer to all this.
We usually use 0.05% tripisin-EDTA, or dilute 2.5% into 0.05%. Anyway, enzyme digestion has some damage to the cell surface, where the antibody located.
I agree with what you all said.
but for EDTA, it does has some effect on cell detachment as it is what we are doing now.
we do not use trypsin as it might affect the downstream experiments, so we use EDTA to detach cells and always have cells detached after several minutes' incubation.
Does anyone know how Accutase works? I understand that it has proteolytic and collagenolytic activity; however, what are the specific enzymatic targets/mechanisms for cell detachment via Accutase?
As mentioned in this thread, trypsin is a serine protease that cleaves cell adhesion proteins at arg and lys residues; however in receptor-targeting studies, it seems dangerous to use trypsin with the risk of unwanted cleavage of target surface receptors (or any surface-exposed proteins with arg or lys residues). Accutase claims to be less harmful than trypsin (I assume this means it has more cleavage specificity), but I would really like to know what is being cleaved.
Thanks
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.
Rhombus,
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 ?
Thanks !