to many dead cells for FACS - how to prepare "sheroid" cells gently for FACS (Jan/17/2008 )
Hi you all,
I wonder, if someone could help me with following problem:
I have so called "spheroid" cells and I want to do some Facs analysis with them. therefore I have to dissociate them into single cells and that's the problem because after dissociating the spheroids mechanically by using versene (10xPBS + 0.5M EDTA) nearly 70-80% of the cells are dead or the cell membrane is damaged. I try to reduce the number of dead cells by only using very small spheroids but that was not effective enough. still 70% dead cells.
What else can I try, because otherwise I can forget my plans with FACS analysis?!?
Please help me! cheers, ceegee
how much time is there between your versene treatment and the time you do facs analysis (might be that your cells are in absence of nutrients for too long so they starve and go death? Do you fix your cells?
I resuspend the spheroids in 3ml versene for 5 minutes, dissociate them during the 5 minutes with a 5ml plastic pipette and after the 5min add 5ml medium. then I go to the lab with the FACS and the analysis is done right away. for the way I may need 10-15min. but I think it's enough to add new medium to the versene to stop the reaction and give the cells new nutrients. I don't think that's the reason for so many dead cells. I think it's more the problem of the preparation...
so maybe someone has a clue how to prepare them into single cells without make them dying?!?