> Hi Patty,
>
> In our hands we use a routine staining buffer (PBS) that includes Fc
> block in all staining and washing steps. I use 2.5% total protein in
> the block, 1% BSA,1% FBS and .5% nmIg. It can be pricey if you use at
> the recommended concentrations we are used to so we grow it up with a
> hybridoma Ig spitting cell line from ATCC ; MOPC-31C for staining
> mouse lymphocytes at 500ug/ml. The Bible for my generation was and is
> Harlow and Lane, Antibodies-A Laboratory Manual, Cold Spring Harbor
> Press for all this kind of stuff..
>
> Gene Pizzo
>
> Manager, Flow Cytometry Facility
> UCONN Health
> Farmington, CT. 06032
> http://flowcytometry.uchc.edu
> 860 679 7567
>
> -----Original Message-----
> From: Patricia Lovelace [mailto:patricia.lovelace@sbcglobal.net]
> Sent: Tuesday, July 12, 2005 6:38 PM
> To: Cytometry Mailing List
> Subject: Fc receptors on Embryonic Stem cells?
>
> Dear Flowers,
>
> Would anyone happen to know if Fc receptors are expressed on human
> embryonic stem cells or mouse embryonic fibroblasts? A colleague is
> having some non-specific staining background and is wondering if Fc
> receptors should be considered as a possible culprit.
>
> As far as Fc blocking is concerned, I have searched and found many
> differing opinions as far as what to use: 2% up to 20% serum, IgG
> 200ug/ml up to 10mg/ml, and , in the mouse system, the monoclonal
> antibody directed against the Fc receptors. Can anyone advise me as to
> how to choose a method of blocking? Trial and error? Is there an
> upper limit as to how much serum or IgG you can add to the
> staining buffer (ie, possible adverse effects)? What is the latest
> thought on Fc blocking when staining intracellular markers following
> surface staining? Does one need to re-add the blocking reagent with
> each step?
>
> Thanks for any advice. I know that the topic comes up a lot on the
> list, just wanted to revisit it.
>
> Patricia Lovelace
> Manager, Flow Cytometry
> Geron Corporation
> 230 Constitution Drive
> Menlo Park, CA 94025