7 replies to this topic

[sea_liu]

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.

[Sprag]

Yes, you can use EDTA only, but it's usually not very efficient, that's why you have the combination.

[jeffreybarrett1]

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.

[sea_liu]

thanks!

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!

[caro]

Hi
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

[sea_liu]

hi  caro:

what is the result for epithelail cells dissociation using PET?

what is the first one for(10% Polyvinylpyrrolidone solution )?

thank you!

[caro]

I find that it is good, polyvinylpyrrolidone disperses the cells to avoid cell clumping.
Regards Caro

[themoon]

jeffreybarrett1, on Dec 7 2004, 09:47 AM, said:

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.

What concentrations of each would not cause damage to the cell surface proteins?