FROZEN CELLS
reb4@NIOBBS1.EM.CDC.GOV
Thu, 04 Mar 93 12:32:00 EST
Here is a response from Jan Nicholson re: frozen cells
Ray,
We looked at frozen cells years ago and published that data in Journal
of Immunological Methods, vol. 73, pages 29-40, 1984. It was a small study,
but we found no differences in % results from frozen cells compared to fresh.
Additionally, we looked at number of antibody-binding sites on frozen cells
from one donor as part of a validation study, and found that they were stable
in at various days (not months or years) after being frozen. The variability
we found was no greater than that from doing replicates on fresh specimens.
That is published in Pathobiology, vol. 59, pages 57-61, 1991. For years we
did studies using frozen cells. The problem is that when they are frozen,
you must freeze them appropriately in the right media, then thaw them
quickly.
Our procedure is to resuspend a cell pellet in cold FCS at twice the
concentration you want in the end. You then add an equal volume of cold
RPMI-1640 that contains 20% DMSO (v/v) dropwise so that the final
concentration of FCS is 50% and the final concentration of DMSO is 10%. This
is all done in the cold, then aliquotted into cold Nunc vials. They can then
be frozen in a controlled-rate freezer, or as we do with double bookbags. We
wrap the vials in tissues, put them in a small padded bookbag, tape it shut,
then put it in another larger bookbag, and put the bookbags in a -70 C
freezer for overnight. The vials are then put them in the vapor phase of a
liquid nitrogen freezer. The recovery is basically the same as that with a
controlled rate freezer. When we thaw, we take the vial out of the freezer
and put it on ice to transport to the lab. It is then put into a 37 C water
bath and swirled until the last ice crystal is almost melted. The vial is
then transfered to the ice until dilution with media. For dilution, we
pipet the contents of the vial into a 50-ml conical tube then add dropwise a
solution of warm (at least room temp) RPMI-1640 with 10% FCS to 10 volumes of
media to volume cell suspension. We then add another 20 ml RPMI-FCS and
spin at about 200 - 300 x g for 15 minutes. After another wash, the cells
should be ready. Our viability is usually greater than 95% for healthy
donors, and our recovery varies from 50 to 90%. Losses do not appear to be
selective, though of the mononuclear cells, monocytes appear to be the most
hardy.
Good luck,
Jan Nicholson
CDC
------------------------------------------------------------------------------
REPLY FROM: Nicholson, Janet FROM: Biagini, Raymond E. (Ray)
FORWARDED FROM: Biagini, Raymond E. (Ray)
DATE: Feb 25 02:06:35 1993 -00:00 relative to GMT
FROM: [A=ATTMAIL; O=attmail; DDA.TYPE=ID; DDA.VALUE=internet(b)ux1.cso.uiuc.ed
u(b)auger]
SUBJECT: frozen lymphocytes
IPMessageID: internet0560206360
TO: [A=ATTMAIL; O=attmail; DDA.TYPE=ID; DDA.VALUE=internet(b)flowcyt.cyto.purd
ue.edu(b)cytometry]
PRIORITY:
Can anyone give me references to working with cell surface antigens (i.e.
CD4/CD8) on frozen/thawed lymphocytes? Is it possible without introducing
artifact? What kind of conditions (freezing/thawing) must be met to make
the procedure possible?
I imagine that there have been some published reports on the feasibility
of freezing isolated lymphocytes and then staining them with various
monoclonals, but quite frankly, if someone can send me their advice, it
saves me from going to the library!
I would appreciate any and all comments. Thanks.
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Julie A. Auger, Director 217-244-0559 voice
Flow Cytometry Facility 217-244-0466 fax
University of Illinois at Urbana-Champaign auger@ux1.cso.uiuc.edu
231 PABL, 1201 W. Gregory, Urbana, IL 61801
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
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