Cell debris here, there, everywhere!? - (Jun/28/2011 )
I use a stably transfected CHO cell line and trying to thaw these cells from liquid N2. I follow all the protocol - quick thawing at 37C, centrifugation for removal of DMSO - but the next day I see cell debris like everywhere. It fills the entire plate and the cells are rounded up and definitely appear to be undergoing apoptosis. I know freezing probably didn't make them happy but I followed the proper protocol there as well - media with 20% FBS and 10% DMSO with freezing at 1C per min. using an appropriate freezer container at -80C and dumped them in liquid N2 following day.
Biggest worry is i have used up all the cell stocks we got from ATCC and all the stocks i made from them seem to disintegrate forming cell debris every time i thaw an ampule. These are precious cells - stably transfected - we can't get them back unless we do cloning again. Stumped !!!!?!?!?!?!
I normally used T-75 for thawed cells with 12ml media. We ran out of T-75s so i now use 10cm petri dishes with 8ml media. I have also tried T-150 with 20ml media. I see cell debris in all 3 cases
I don't think its contamination as i don't see any bacteria or the media doesn't change color or anything until and unless all cells die and lift off the plate. No one else has a similar problem although we share FBS and P/S, the hood, incubator etc. I'd really doubt it was mycoplasma coz my cells don't even last 12 hrs before dying out completely. It seems to be a classic case of them not being happy fr some reason.
right after you thaw the cell, you should check the viability of the cell and count the number of viable cells
then you should decide which culture flask you should use.
T-75 might be too large, if majority of cell are dead. try T-25 or smaller flask.