gld lymph nodes

Kenneth Frauwirth kfrauwir at midway.uchicago.edu
Fri Feb 19 02:31:42 EST 1999


In article <B19E4F226C2DD211BE8E0008C7FAB88F079FE5 at BHQROC1EX1>,
Chen, Peter <ChenP at USA.REDCROSS.ORG> wrote:
>
>Hi, I was wondering if anyone knows a technique that eliminates
>double-negative T cells while enriching for B cells from the lymph nodes of
>gld Balb/b mice. I'm experimenting with magnetic sorting but there are few
>ideal antibodies that would target DN T cells with high efficiency and some
>antibodies (for instance anti-TCR) are not made in rat (the magnetic
>particles are conjugated to anti-rat IgG). This technique will be an
>enriching step before FACS cell sorting but I need to enrich a target
>population that only constitutes about 2% of the total. Any advice would be
>greatly appreciated!

If the antibody species is the biggest problem, perhaps your best bet is
to just get magetic beads conjugated to avidin and use a biotinylated
antibody.  There are also some commercial cell-enrichment kits that work
very well.  I have had great success with the T cell enrichment setup
from StemCell Technologies, and I am sure that they sell a B cell
enrichment version - these use cocktails of antibodies to get very good
purifications (I typically get >95% pure T cells from normal spleen).  I
have also done serial separations to get CD44-high and -low populations of
purified T cells.  According to the brochure, the original design of
the system was for the purification of hematopoietic stem cells,
so it is supposed to work for the enrichment of very rare cell
populations.  The downside is that it is pretty expensive.

Ken


-- 
Ken Frauwirth (MiSTie #33025)  kfrauwir at midway.uchicago.edu
Gwen Knapp Center for Lupus and Immunology Research
University of Chicago
http://www.ocf.berkeley.edu/~frauwirt



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