I would like to measure cell surface IgG in human culture of B cells. I have culture of human peripheral blood mononuclear cells (PBMCs) that were immortalized with EBV (Epstein-Barr Virus) and cultured in medium supplemented with 10% FBS. For IgG surface analysis we stained cells with anti-human IgG antibody (Miltenyi, 130-119-772, APC, host mouse), following the provided protocol. Quite high percentage of PBMCs were positive for surface IgG (14%). Assuming unspecific binding we used human FcR blocking reagent (Miltenyi, 130-059-901), according to the provided protocol. With FcR blocking the results were almost completely negative for surface IgG.
- Do I have to use blocking reagent in this case or is it sufficient if the cells are cultured in medium supplemented with FBS?
- Does anyone have experience with staining surface IgG and cross-reactions with FcR receptor?
Hi,
My suggestion to you regarding negative results with FcR blocking reagent is that maybe the concentration of Ig in serum is too low for sufficient blocking, and therefore it's recommended to use purified IgG.
Fc block binds to fc receptors. I doubt this is the issue. recommend targeting igm rather than igg on the b cells. The virus does not induce class switching on infection and most circulating b cells will be Igm +. Also, bcr components can down regulate following ebv transformation. If the cells have been passaged for a long period of time, this may block surface expression of bcr.
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