HTC116 cells detaching !!!! - (Apr/01/2011 )

Hi...

Has anyone had this problem before???
My HCT116 cells keep detaching from the T75 plate. I usually wash the T75 with PBS before adding tryp-edta for passaging, but since last week some of them are being literally washed out by the PBS. It had never happened before.
Could it be a sign of over confluency in the plate? I passage them twice a week and I need to do a 1:22 dilution, because if I do anything more concentrated than that the plate is overconfluent in no time. ATCC recommends 1:8 passages but it has never worked for us.
Are my passages ok or should I do less diluted passages more frequently ??
Thanks for your help.

Isolda

Is the PBS OK - i.e. correct pH? It could be that you are damaging the cells with the wrong pH and they are lifting off in response.

Your PBS doesn't contain EDTA (by accident)?

How long have you had the cells up?

Would (do) you still use them for experiments even though they are behaving differently to normal cells?

I have worked with HCT116 cells, and this has happened to me before. I think you're right that it has to do with overconfluency. They grow like wildfire, and they tend to grow on top of themselves, making them top heavy. Btw, if you have a good method of dissociating them (I just can't seem to get a single-cell suspension), let me know!