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im currently working on podocyte cell lines. protocol recommends use of accutase to detach adherent cells. can this be replaced by trypsin?

I want to do some functional assays to test integrin AVB3 activity on the surface of some cancer cells. I used to use accutase to gently detach cells from the surface for these assays and it worked great. But now our lab only has cell stripper. I'm just wondering which would be better to use for studies on integrins. I know cell stripper works like a chelator that dissociates ions from integrins and let integrins release their substrate. Accutase on the other hand seems to work like protease and collagenase so it's more similar to trypsin but only more gentle? Does anybody have experience using cell stripper for integrin assay? I think in theory it should work just fine but want to make sure. A few papers I've read all used accutase for some reason. Is there any advantage of accutase over cell stripper?

Thank you in advance!

FBS includes various protease inhibitors paticularly α1-antitrypsin and can inactivate trypsin. Can FBS also inactivate accutase?

Which one of 3 solution above is the best to be used for cell culture? Anyone? THANKS :)

Does anyone knows the mode of action of accutase? It is known that it is gentler cell detachment method compared to trypsin. There are literatures stating that they are a mixture of proteolytic and collagenolytic enzymes which were extracted from crustaceans. They also degrade E-cadherins. But why is accutase gentler compared to trypsin? In what way do these enzymes (trypsin and accutase) work differently? (I don't know why but there is very few information about accutase mode of action available on the internet). Thanks.

Benny Burn

Hello, I've been using Accutase recently to detach my adherent cells. I was previously using trypsin-EDTA. According to the manufacturer's indications, Accutase can be stored long-term at -20 and short-term at 2-8.

When at 2-8, do you store the whole bottle or you create aliquots that can be readily used?

Also, do you thaw at 4 or thaw in a 37 degree water bath?

Thank you.

Process development. Is this enzyme inactivating/digesting itself when kept at room temperature for long period of time (ie. 6-8h) , such as seen with trypsin? any relevant reference appreciated. Best

I have converted iPSC to cardiomyocyte and I would like to make functional assays to cardiomyocyte so I need to passage them. If I used accutase for this passage, does it cause any damage to cells or does it work to deattach the cells?

I am stuck with the dilemma of my SH SY5Y cells looking like they are clumping and growing on top of one another with some space around them. They are not spreading around very evenly like some of my colleagues described. Funny enough, one colleague of mine is using 0.2% trypsin, another using tryple and another using accutase all subjecting to SH SY5Y cells and they hardly have any issues like mine.

Note that I have tried about 10 rounds of splitting from a revived batch from 10 separate cyrotubes and most of them ended up curling up like a ball and float - which I believe signifies cell death.

I have tried various concentrations like 2 mM EDTA, 0.02% Trypsin in 2 mM EDTA, 0.2% trypsin in 2 mM EDTA, 0.2% trypsin in 0.53 mM EDTA (this one following ATCC protocol) and even tryple and they still show signs of clumping after that.

These are the questions that I need answering:

1. Should I try accutase as I read its similar to EDTA but much less harmful from the rest?

2. The only suspected issue left is that after lifting the cells from the T25 flask, and then centrifuging them, I did not do a thorough rinse of the T25 flask with either PBS or with media in FBS to either further neutralize the trypsin/EDTA/tryple or remove any left overs of those cell adhering solution. Is that the only problem that I have?

3. Before trying out different cell adhering solutions, I used to use 2 mM EDTA alone and I used flask knocking to adhere the cells before centrifuging after subjecting it for 5 minutes. On the other hand, many have told me that when they were using their choice of the cell adhering solution, they wait until the cell lifts up and becomes spherical and they didn't need to knock. Should I follow suit and exposed the SH-SY5Y cells longer to EDTA until it becomes spherical?

4. Among the solutions listed in the title, which is more harsh to least harsh that they would reduce the chances to lyse the membranes of the cells when exposed too long? And do you recommend other types of "cell lifting" solutions other than the ones listed above?

Would be great to get your feedback as I understand from many of the different researchers that ppl used different cell adherent solutions with different timings and still get the result they needed. Thanks

I am trying to dissociate primary neurospheres (mouse), which are cryopreserved in Liq.N2. I followed the standard procedure given by the supplier and the same procedure was followed in many publications. to my wonder i observed that all the neurospheres are getting clumped and Trypan Blue shows all the cells were dead. In continuation, i have diluted the accutase 10 times and i could see improvement in cell viability..my question is why every one else are able to dissociate cells in direct accutase and why i am not able to do it..I wonder why? as per my knowledge no publication mentioned about diluting the accuatse..please clarify my doubt..thanks