>Date: Mon, 5 Feb 96 10:03:13 CST
>From: Tom Waldschmidt <Waldschmidt@tjwald.path.uiowa.edu>
>Subject: B cell subsets
>To: gebhard@aecom.yu.edu
>
>Dear David,
>
>We have been focusing on identifying murine B cell subsets for some time, and
>have devised 3-color protocols to separate follicular, marginal zone, and
>immature B cells in a resting spleen. This protocol is based upon the use of
>B220, CD23, and HSA as markers. For a review, I would refer your user to:
>METHODS: A companion to Methods in Enzymology 8:3, 1995. In an activated
>spleen, plasma cells can again be detected by a combination of markers. The
>suggested combination would be B220+, Mel14-, and PNA+. I would point out
that
>PNA staining is a titration-dependent phenomenon, and care must be taken when
>using this reagent. Memory B cells in the mouse are a particular problem,
given
>their extremely low frequency and lack of unique surface markers (see PNAS
>84:1379, 1987). The safest marker is surface Ig of a switched isotype (IgG or
>IgA). Again, this is tricky since most monoclonal antibodies directed against
>these isotypes recognize soluble and not surface forms. A very good polyclonal
>reagent (with no IgM, IgD, or light chain cross reactivity) is the best bet.
>
>Best regards,
>
>T. Waldschmidt
>University of Iowa
>
>
>
Dave Gebhard
Director FACS/ Oligonucelotide Synthesis Facilities
Albert Einstein College of Medicine
1300 Morris Park Avenue
Bronx, New York 10461
718-430-2724/ 3573