HEK 293T cells - problems getting proper attachment. - (Apr/14/2008 )

Hello,

I am working with HEK 293T cells, and I am having some serious problems getting them to attach to the culture flasks and dishes i use. There seems to be no pattern in the way they behave, sometimes they attach and grow and sometimes they don't. It would seem they prefere NUNC products, but not always. To grow them, I am using glutamax media with glucose, pbs and streptomycin/penicillin. I wash them in 1xHBSS buffer, tryptinate them for 2 minutes, inactivate with media and centrifuge them. After that, I split them into new flasks/dishes.

Has anyone experienced problems getting this particular cell type to attach and if so, how did you (or didn't you) solve it?

-elpollodiablo-

hi there!

these cells are a little touchy.
be sure to treat them nicely, eg. medium and buffer really needs to be warm enough, never pour the medium directly onto the cells. if you need them to attach good, you can coat the plates with poly-l-lysine.

squishy

-squishy-

Thanks! Ill try that out - anyone else has any suggestions??

-elpollodiablo-

They are a bit sensitive but if everything is okay with them they should attach ON. Or maybe something is wrong with that batch of medium??

I used to keep them in DMEM, 10%FCS, 1% PenStrep, 1%L-Glu and 0.1% beta-mercaptoethanol. They detach quite easily. I just add 1 ml trypsin, distribute evenly on the plate, aspirate off. The cells start already to detach. Plate them as usual.

-zek-

Well, I've tried to change media, but it was the same! I'll try to keep it warm from now on, see what happens!

-elpollodiablo-

Did you get any cell clumps? If so, it is possible that you did not resuspend the cells into single cells suspension when you plated out your cells.

-Franz K.-

my colleague used to call them the jellyfish cells, because the whole plate of transfected cells would suddenly lift and swim around like a jellyfish. not much help but just to let you know that these cells are a bit problematic sometimes

-Lusen-

These cells are quite finicky. Be very careful while washing with PBS. Any harsh handling will make the cells to come off. I grow them in DMEM+10%FBS, in Greiner flasks. Add PBS on sides of the flask tilt flask gently twice, aspirate off and trypsinize for 2 min, and quench with media. I have not faced 'attaching' problem even when I grow them in 35mm, 60mm and 90mm dishes for transfections.

-Calvin*-

QUOTE (Lusen @ Apr 25 2008, 04:31 PM)

my colleague used to call them the jellyfish cells, because the whole plate of transfected cells would suddenly lift and swim around like a jellyfish. not much help

but just to let you know that these cells are a bit problematic sometimes

haha.. thats what i call them too! Anyway, i'm working with 143B osteosarcoma cells now, and im starting to see the jelly fish thingy again!... i guess it happens when cells clump together when plating..
cheers

-jingz-

QUOTE (jingz @ Jun 9 2008, 07:15 AM)

QUOTE (Lusen @ Apr 25 2008, 04:31 PM)

my colleague used to call them the jellyfish cells, because the whole plate of transfected cells would suddenly lift and swim around like a jellyfish. not much help

but just to let you know that these cells are a bit problematic sometimes

Sometimes if you don't change the media often (~ every 2-3days) some will get floating and maybe dying. But the majority will still attach, so we never mind about that. And we never centrifuge the cells when we split, just make sure they are nicely suspended and no clumps formed would be better for the problem.

BTW we us MEM media with 10% of serum, 1% of L-glu, 1% non-essential amino acid and 1% pen/strep. And the cells are always growing well unless you got some problem with the liquid nitrogen and they are all dying.

Good luck!

-layilayo-