Hi Jakub, I didn't get the original post, but I wanted to let you know about
another method of Bcell stimulation that I've used before- polyclonal goat
anti-IgD. It was originally developed by Fred Finkelman (i think) and I've
used it in murine models in vivo systems with lots of luck- it should work in
vitro if you can find the antibody. Interestingly, this type of stimulation
is T cell independent, although it prefferentially activates naive B cells.
mbdxm at my-deja.com wrote:
> Hi Jakub,
>> Purify your B cells first then use PMA (phorbol
> ester) 10 ug/ml and ionomycin 1 ug/ml.
>> LPS should have worked but its not specific to B
>> As for monkey anti-IgM ( anti-mu should be better),
> try the antibody resource pages on the web there
> are many usually as part of a company site.
>> You might need to crosslink the anti-mu to get a
> mitogenic signal. Even better if you stimulate your
> cells with IL-4 and anti-IgM.
>> In article <382A22EC.D1A9E24F at stanford.edu>,
> Jakub Regieli <jregieli at stanford.edu> wrote:
> > I'm trying to achieve polyclonal activation(and proliferation) of
> > B-lymphocytes in vitro.
> > I'm having difficulty finding the right stimulus (mitogen or MAb) to add
> > to culture, to stimulate specifically B lymphocytes.
> > I tried Lipopolysaccharide (Sigma E.Coli 055:B5), and Staphylococcus
> > Aureus Cowan I (Calbiochem, product name Pansorbin).
> > None of these worked, as they should according to literature. I might be
> > using the wrong clone or origin of the stimulating agents. I'm working
> > with peripheral blood of cynomolgus monkeys.
> > I'm not sure where to obtain anti-IgM antibody that would crossreact.
> > Does someone have a suggest where to search for the correct mitogen?
> > I'd appreciate any help/directions you could give me.
> > ------
> > Jakub J. Regieli
> > Transplantation Immunology
> > dept of Cardiothoracic Surgery
> > Stanford University
> > lab 650 - 498 - 6414
>> Sent via Deja.com http://www.deja.com/> Before you buy.