Cell pellet after freezing - (Dec/10/2008 )

Hi all,

When I was transferring my cells to the N2 stock today I noticed that the cells (keratinocytes and fibroblasts) had formed a pellet at the bottom of the vials. I used a standard protocol: pellet cells, resuspend in medium + 10% FBS + 6% DMSO, aliquot, store in isopropanol container -80 C, and after 1 day transfer to N2. I have not noticed such a pellet earlier. Do you think that the pellet will have any effect on the viability of the cells (also after thawing)? And if so, do you have an idea how to prevent this?

Thanks in advance
David

I've never heard of it of seen it, but that doesn't say a lot!
If you're worried about cell viability after thawing the cells do a cellcount with Trypan Blue or another live/dead dye so you can see if you cell viability is really bad. You'll always loose some cells, but you shouldn't have a lot of dead cells in your sample.
The protocol seemed fine to me, only I've always used 20% DMSO, 40% FBS and 40% medium. But I've seen different freezing medium to...

20% DMSO seems too high for me
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