Trypsin-Free Detachment of cells using EDTA - (Jun/20/2006 )
I am trying to detach T84 cells without trypsin, however these cells are especially hard to lift. Can anyone tell me how long cells can incubate in EDTA before it harmful effects set in (ie apoptosis, membrane damage, etc)? So far 10 minutes is not enough to lift all the cells.... Thanks in advance.
Why don't you want to use trypsin?
if u need to detach cells, i have a protocol with citric saline.
I am interested in studying cell surface proteins via flow cytometry, which trypsin would eat up. I am very interested in your citric saline protocol... You can email to me if that's easier. Thanks so much!
check ur PM
thanks so much scolix - i will try it.
can u send citric saline method for detaching. i am also intrested.
If possible, I too would be interested in a copy of the non-trypsin based protocol. Thank you in advance if you may offer any assistance
Me too, pleasssseee. Thanks.
Here are two different approaches I have been recommended:
1) EDTA only Method:
Make a solution of 1mM EDTA (can be increased all the way up to 10-15mM EDTA if your cells are extremely adherent). The EDTA solution acts to chelate calcium. Flood flask with the solution and incubate for 1 minute. Remove most of solution (not as dangerous for cells to sit in EDTA as it is to sit in trypsin) and allow to incubate at 37degrees for 5-10 minutes. Time may be increased if necessary.
I found that the EDTA solution at 10mM worked well for 10 minutes with Hep2 epithelial cells, but I had difficulty with clumping for my T84 cell line. Normally this would not be terrible, but for flow cytometry it is very problematic.
2) Citric Saline Method
For 500mL 10x solution: 50.3 g KCl, 22.06 g Sodium Citrate.
Flood flask with prewarmed citric saline (1x) and incubate at 37 degrees for 4 minutes. Remove cells be gently tapping or pipetting up and down several times.
I have not had the chance to try this yet, so please let me know how this works for you!
Hope that helps...