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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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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web