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Lin-negative for murine bone marrow cells refers to the standard
combination of antibodies against CD4, CD8, B220 (CD45R), Gr-1
(Ly-6G), Mac-1 (CD11b), and Ter119. There are many suppliers of each
of these, but Pharmingen is the only one I'm aware of that can supply
them low endotoxin/azide-free (my experience, however, is that the
azide does not effect stem/progenitor cell viability during an
immunomagnetic depletion- we can routinely get >80% of sorted cells
to grow in colony assays after a sort). Be careful; there is some
controversy regarding the presence of CD4 on stem/progenitor cells
and some investigators prefer to substitute CD5. My own experience
shows no depletion of stem cells with CD4 antibody. It's probably not
a good idea to deplete with Thy-1 since mice with the Thy-1.1 allele
express the antigen on almost all stem cells (see Spangrude, Annu.
Rev. Med. 45:93-104). Also, stem cells in fetal liver are Mac-1
positive, so avoid this one if using FL. If you are planning to
deplete with magnetic beads, you should be aware that it's almost
impossible to get thorough depletion and you may be left with your
population being 50% or so lineage-positive. The best way to deal
with this is to use a second step goat anti-rat-APC or -CyChrome (all
primary Abs are rat IgG) or biotin-labeled primaries with the above
secondaries to eliminate residual cells on the FACS.
Good luck,
Steve Neben
Genetics Institute
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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