Kalpit A. Vora
K_Vora at lac.jci.tju.edu
Tue Apr 14 15:56:14 EST 1998
In article <Pine.GSO.3.96.980413132953.29697B-100000 at dilbert.ucdavis.edu>,
"L.A. Pike-Nobile" <ez063669 at mailbox.ucdavis.edu> wrote:
> I am getting extremely variable rates of viability from my B cell lines
> when I thaw them after storage in liquid N2. I freeze them in 50% media/
> 30% FCS/20% DMSO. I freeze them gradually (I use on of those little
> ethanol-jacked freezing things that claims to give 1oC/hour) and I thaw
> very rapidly (37 water bath) and rinse them thoroughly.
> AND YET--I still get some vials with less than 1% viability while others
> are fine. Some lines are better than others, but I see this problem even
> within the same line, frozen at the same time. I'm worried about
> completely losing some of my more fragile lines.
> Are there any tricks I should know about?
We routinely freeze our hybridoma in fetal calf serum containing 5%DMSO
and nothing else. Also We pellet the cells resuspend in the above freezing
medium and leave it overnigt at -70 before transfering to liquid nitrogen.
It can remain at -70 for 3-4days without effecting the recovery. Also try
and freeze the cells when they are young and growing in exponential phase.
I donot see any problem in the way you thaw them out.
Hope this helps.
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