janjan wrote:
> Does anyone know of a technique (if one exists) for effectively freezing
> and thawing T cells-- and getting them to proliferate after thawing?
>> Thanks.
> Janet
I routinely froze mouse T cell clones 2-3 days after stimulating them with
irradiated APC and peptide. Just use 5% FBS-Click's (Irvine Scientific) with
10% tissue culture grade DMSO (Sigma) as your freezing medium. Important thing
is to LSM your T cells, then wash a couple of times with 5% Click's, resuspend
the pellet, put the tube on ice and drop-wise add ice-cold Click's/DMSO with
gentle shaking. I just packed a beaker with ice, plunged the tube into the ice,
added my freezing medium while swirling the beaker (and consequently the
cells). Once you've got them to 10^6/ml, aliquot into cryovials, put them in a
box with a lid and freeze them at -70 overnight. Then transfer to liquid
nitrogen. To be safe, pull out a tube from LN2 after overnight and check for
viability. Should be >90% by trypan blue, and should be able to put them up
with APC and peptide.
