I'm trying to develop the functional assay for B-lymphocytes. I'm going to treat C57Bl/6 mice with compound of interest and immunotype cells in the whole blood and spleen by FAX (to compare with DMSO-treated animals). I'm wondering, if any functional assays for B-cells in the whole spleen culture is possible to do? I found papers for stimulation of B-cells with anti-IgM F(Ab')2 (10ug/ml), or anti CD72 K10.6(10ug/ml), or both, or anti-CD40 (10ug/ml). How specific are those simulators? PMA and ionomycine are not specific a priory.
It's strange, that people used anti-CD72, because it suppose to be the inhibitory one.
Hi Liliya,
it depends on type of function you want to test.
However, if you need to stimulate them, use either CpG-B or a combination of anti-IgM F'(ab)2, IL-21 and anti-CD40. You also may want to consider to isolate the B cells first by negative selection using magnetic beads, so that you can 100% sure that only the stimuli are influencing the B cell function you would like to study.
You could use Mitogens like bacterial lipopolysaccharide oder lipoprotein, anti-IgM antibodies and CD40. Using LPS is cheap and has been done for almost half a century .
The use of an anti-IgM target mainly the naive B cells, as well as the anti IgD, but also the Bm2 which are hypermutated B cells.
The use of CpG mainly targets memory B cells, which must be purified to improve the score, otherwise the macrophages will be the first stimulated, and when stimulated it is not good at all.
LPS is not a good solution for a lot of reasons. SAC associated with Il4 and / or Il10 is more suitable.
Activation via CD40, or CD154, is the best solution. You can use an anti CD40 antibody, or a CD154 protein. But the best way is the CD40 system associated with fusion with SP2o, especially if you are looking to analyze the antibody response of the mouse.
But be careful, never do a final boost otherwise you will lose all the B cells of interest that are in the spleen.
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