[Histonet] marker to measure if sorted or FACSed cells are contaminated?

koellingr <@t> comcast.net koellingr <@t> comcast.net
Sun Jul 8 22:02:20 CDT 2007

If for CD11, you mean something like CD11b, CD11b and F4/80 would cover monocytes, granulocyte, macrophage lineage type cells and but there are other antibodies available that would show cells from that kind of  linage on earlier BM cells better.  Both do work in  FACS, Westerns and IP as I've used them.  But they won't touch your B-cell population for which you might want to try CD20 or CD45R (the B220 A-exon restricted isoform component of the CD45 complex).
Also, picking out bands on a Western from (minimally) contaminating cells that have been lysed along with your target population can be problematic.  Could you sort (flow) them?  Magnetic beads are great and fun and doable and quick, but a cell sort I think would be better.  Your e-mail appears to be NIH so there must be high-speed cell sorters in abundance around.  And with the abundance of mouse BM cells, it wouldn't be a long sort.  And anyone who sorts would know the antibody Ter119 which is a fantastic MOUSE erythroid marker from early to late erythroid cells.  Sorting with CD11b, F4/80, B-cell markers and Ter119, gated properly, will give you some demonstrably pure erythroid populations and you would't have to search for contaminating cell lysates in Westerns.  And for the IHC people Ter119, does work great by IHC, on mouse.
Ray Koelling
Phenopath Labs
Seattle, WA

-------------- Original message -------------- 
From: scoop <@t> mail.nih.gov 

> Dear Histonetters, 
> I'm sorting mouse bone marrow cells (using magnetic beads). I would 
> like to do a western on my sorted cells to determine whether the 
> erythroid cells are contaminated with macrophages or B-cells. Does 
> anyone know of an antibody to a mouse bone marrow macrophage marker 
> and a mouse bone marrow B-cell marker that I can use for this 
> purpose? I was thinking about F4/80 and CD11 but I don't know if 
> they work in a western. 
> Thanks in advance! 
> Sharon 
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