Arnold: 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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