How to digest my cell line/strain to a single-cell suspension? - Help! (Sep/03/2007 )

I set up a cell line.
But I've never digest the cells to a single-cell suspension
They always clump together, or are just like a string of beads.
After passaged to P11, I did a single cell cloning by serial dilution.
Then I set up a cell strain.
But like father, like son, you know.

Will someone please give me a hand?
Thank you!

PS: I always digest them with T/E(Gibco),
and one time I adjusted the EDTA concentration to 5mM(Commercial products are about 0.5mM?), but failed.

Hi,

Don't over trypsinise your cells! you are probably leaving them in trypsin for too long, 3-5 minutes maximum is what you should be using, after this they will clump and you will never be able to get a single cell suspension. I ususally trypsinise until I can see a few cells just lifting off, then tap the side of the TC vessel hard untill all the cells are floating, after this add the medium and pipette up and down a few times to ensure most of the clumps are broken apart and that should do it.

Note that some cell lines will need less than 3 minutes!

you want to swith from attached to suspension cells?
or just suspension cells ?

Well, in both case, i think of joklik's medium. Hela S3 cells are attached cells in standard medium (DMEM, optiMEM or RPMI) and goes in suspension when cultured in joklik's. I think it's because it's calcium chloride free.
So i would try this to culture our cells, which may decrease contacts between them.
Joklilk's medium is available in powder from eurobio.

Thank you two

Bob: Maybe you are right, I do trypsinized the cells for a long time during the development of the cell line.
Because they are very hard to detach from the cell culture dish.
You said "never". So my hope is an impossible mission, isn't it?

Fred: I don't want switch to culture suspension cells.

ok i wqas wrong. But the fact you always get clumps is not fatality. pipetting up and down allow to detach cells from each others.

What you ma do : from a plate, resuspend well your cells and pipett 5 10 and 25 µl of that cell suspension and put it in 10cm plates. You'll see clones appera after a little while.
Second possibility, a FACS can do 1 cell per well in 96 well plates for you