B cell proliferation assays - (Mar/24/2001 )

Well, I will try and keep it short. I am isolating B cells from tonsils (teasing the cells out, Ficoll and then AET-treated sheep red blood cell rosetting). The purity of the final population by FACS (CD19/CD3) is about 85-95%.I stimulate the cells with anti-IgM (used to buy polyclonal anti-IgM coupled to beads from Metachem but they stopped producing it) and a second stimulus (eg. IL-4, interferon). It used to work fine. The person who used to do them left so after a year I tried to repeat it. The positive controls (PMA, IL-4) work like a charm. But stimulation with interferon + anti-IgM shows nothing.1) Is it the cells? (contamination?, macrophages in tonsils?)2) Is it the interferon batch ot the concentration? (tried 3 different interferons)3) Is it the beads? I would appreciate any ideas about some sort of controls I could run or any ideas. (period).

Thanks

1) Check to make sure the anti-IgM does not contain sodium azide2) It sounds like you've tried different batches of gamma, what about antibody?3) try coating the antibody to your culture plate or use higher concentrations soluble4) Has your serum been heat-inactivated? Serum proteases may kill your cytokines.