Re: Freezing cells
Thomas Mchugh (mchugh@zion.his.ucsf.edu)
Mon, 4 Oct 93 9:21:03 PDT
>
> Hello All;
>
> I was told that it is possible to freeze cells in Fetal Calf Serum,
> thaw them out, and run surface markers on them. I have some recipies for
> the freezing media, but I was wondering if someone else has some experience
> with doing this. What are some of the potential pitfalls? Do you see any
> change in the surface antigen expression? What is the concentration of cells,
> and how much can you freeze? The lowest concentration of FCS to cells that
> I have seen is a 50-50 mix. What do you add as buffers? Any info would
> be wonderful. Thanks in advance--robert rainer
>
> rainer@phs.bgsm.wfu.edu
We routinely freeze cells and then after thawing analyze them for
surface markers. We freeze them at 10E6 - 10E7 per ml in RPMI + 10% FCS
(or newborn calf serum) + 10% DMSO. We add the DMSO last and immediately
start to freeze either in a temp controlled freezer or first at -20,
then at -70 and finally in liquid nitrogen. Thawing is done at 37C in a
waterbath and when about 75% of the vial (we freeze in 1 - 2 ml
aliquots) if liquid we then transfer the 1 - 2 mls to 1 -2 mls of warm
(37C) RPMI + 10% FCS and mix. We slowly add RPMI + 10% FCS until the
total volume is 15mls. We spin down and wash the cells once then stain
and run. Works great both for cell surface phenotyping as well as cell
function assays. Exposure to DMSO for extended periods of time while not
frozen is hard on the cells.
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