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I would have thought that freezing and thawing a lump of tissue would
result in all dead cells, making a suspension of fresh cells then freezing
using DMSO and FCS in medium should give you much better viability after
thawing.
If I were you I would use an enzymic approach to making a cell suspension.
collagenase and/or dispase and/or trysin would do the job, keeping your
cells in divalent cation free buffer helps prevent clumping as does a tad
of DNase (10u/ml). As I remember several of the collagenases in the Sigma
catalog are described as suitable for making cell suspensions from liver.
Doing your surface staining, fixing in formaldehyde for an hour then fixing
in 70% ethanol then RNase then PI is the way I stain lymphocytes with
surface markers and PI. Ammonium chloride lysis isn't that harsh and
shouldn't kill your cells. High background staining is not suprising if all
you cells are dead.
I have no experience of liver but make suspensions of cells from gut
tissue, I actually dont use enzymes any more as they changes some of the
markers I was looking at, I use the Dako medimachine, enzymes result in a
better yield and better viability though.
Simon Monard
Aaron Diamond Center
NYC
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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