How to disassociate cells for FACS? - (Nov/28/2006 )

Hi,there,
I'd like to purify cells by FACS with Antibody to E-Cadheriin(this protein mediats cell-cell adhesive junction), after digestion with 0.05%Trypsin-EDTA, or 0.25%Trypsin-EDTA, the cells couldn't be purified because of disruption of E-Cad.However, when disassociated by Cell Disassociation Buffer(Gibco), most cells couldn't become single cells.So How to Avoid of disruption of E-Cad while get the cells into single cells with intact surface protein for FACS?
Thank you for advice!

i've heard of cells being treated with lidocaine to help remove them from the dish. maybe something you could look into.

V

A method I follow for FACS-analysis of membrane surface receptors on U87-cells (which are adherent):
I detach them as usual (with trypsin)
I put them in a tube (not in a culture flask) and wash away trypsin, then resuspend in normal medium. In the tube, cells will re-express the membrane receptors but not become adherent. After 2 hours (might take more time for some receptors, or less for others, never bothered to try to find out the exact minimal time needed, but for my case 2 hours works fine), just label them as you normally would and FACS them.

Maybe this could work? Another option would be to remove them with a scraper (if your cells can handle that)?

Can I use Ultra-low attchament dish for the re-expression of membrane protein?(Cells in this kind of dish don't attach to the matrix). One more question, when you use tube, do you shake the tube or just put it in the hood?
Thank you again

I mildly shake it, but after a short time the cells are to be found on the bottom (they are more dense than medium, hence gravitiy makes them come down).
This hasn't hampered my results.

Maybe you can use ultra low attachment dishes, don't know if they are more expensive or not? (check that in if it's necessary because other ways aren't working you'll have to do it, but I'd first check easy and cheap protocols).

Dear Jerrylee,

What cells are you using.......can they be grown in suspension

Rhombus

mouse embryonic stem cells, which can be cultured in suspension to form embyonic body(EB), or monolayer on gelatin-coated dishes, I have to purify the E-Cadherin positive cell populations from EB or monolayer for my subsquent expeiment.