detachment of my cells?/ - (Jul/30/2006 )

hello all

today i went to do passage for my 239A cells and i found them detached in a visible layer floating in the media????
this is the first time for me to have this kind of problem?
i dont know why?
its has been 4 days since i did passage for them , usually i do passage every 2 days?
may this is the reason?
i took my cells and centrifuge them then discard the media and wash with pBS and centrifuge then do the passage , but ofcourse i found my cells clumped in the new media?
will this be a problem?
thankx in advance

HELP

hello minnie
no i amnot talking about the usual way in passing my adherent cells
i am talking about a strange thing happedend.
the cells detached while it was in the incubator i didnt do anything yet , i got them out from the incubator in order to do the passage and then i noticed this layer?
thats why i didnt do the usual passage technique as if i aspirate the media my cells will be gone with it so i centrifuged the old media with cells in a 15 ml tube and i didnt add any trypsin as its was already detached ?????

Did you wait too long before passing? Did medium appear yellow? Your cells were not very happy. Under this condition, you still need to use trypsin/EDTA treatment, but using less treatment time than usual (otherwise cell dies) to ensure single cell suspension before passing.

Sorry I misunderstand.

I agree with genehunter-1.

Your cell may have overgown, the detach layers may be dead cell. Rinse your cell with PBS to get rid of dead cell, and trypinize your cell as normal.

I hope this may help.

Floating monolayer cells usually mean dead cells. Possible explanations include:

1. Your culture became grossly overgrown. Often cells lift in sheets if this happens (although it is usual to still have some cells attached in patches). This would normally be associated with yellow (acidic) looking media. It can sometimes look a little bit like contamination because the dead cells can make the media look turbid.

2. Your culture became contaminated. Most likely the media would again appear turbid and yellow. If you innoculate a drop of this culture into a sterile tube/flask with new media and incubate it overnight it should also turn turbid/yellow if it is contamination (assuming absence of antibiotics or antibiotic resistance).

3. You killed the cells when you subcultured them, however, as it appears you sub-culture reasonably frequently without any problems this would seem unlikely. I gather other cell cultures in your incubator are fine which would rule out a serious incubator problem.