EDTA only for cell dissociation - (Dec/07/2004 )
now i am culturing epithelial cells which is hard to dissociated by typsin-EDTA in primary culture. i want to know if anyone ever used EDTA only for cell dissociation. any suggestion will be appreciated.
Yes, you can use EDTA only, but it's usually not very efficient, that's why you have the combination.
I would try other cell dissociation enzymes. I don't remember the company, but you could try Accumax or Accutase; go to the following website for information: www.innovativecelltech.com/accutase.html
Both Accumax and Accutase use a mixture of enzymes, which may improve your results. There are also several other cell dissociation products available commercially. I would just try several and see which one works.
You may also want to try a higher trypsin-EDTA concentration and incubation at 37 degrees C. Just make sure not to incubate for too long to harm the cells.
One other note: I have used dispase in combination with trypsin-EDTA to separate spheroids and cell aggregates. You may want to give it a try.
yes, now i am trying dispase for 3h, then use Trypsin-EDTA for further dissociation.
it seems that they did work.
i have another question to ask jeffreybarrett1: when you use dispase do you add culture media to keep cell viability, because it usually takes a long time for dissociation . or use culture media to make diapase working solution?
thank you for your answer!
I use a PET dissociation mix for epithelial cells
PET dissiociation mix:
36 ml Hepes Buffered Saline
5 ml 10% Polyvinylpyrrolidone solution (Biosource International, catalog # 345-020)
5 ml 0.2% EGTA in Hepes Buffered Saline
4 ml Trypsin, 0.25% with 0.02% EDTA solution
Store 25 ml aliquots at -20
what is the result for epithelail cells dissociation using PET?
what is the first one for(10% Polyvinylpyrrolidone solution )?
I find that it is good, polyvinylpyrrolidone disperses the cells to avoid cell clumping.
sorry for such a delay in my response to your question. Dispase won't work in media (unless perhaps it is serum-free, which I didn't try). My dispase solution was made up in HBSS at 10X conc. and then used at 0.5X final concentration with 1X tyrpsin-EDTA solution.
An enzyme such as collagenase will work in media, even if it contains serum.
Hope this helps you or at least someone else.