I would like to stimulate single memory B cells in a short term culture and am wondering what is the point to co-culture 3T3 ms CD40L feeder cells to stimulate single memory B cells. Why soluble CD40L is not easily used instead?
Actually, I am going to harvest supernatant and do further analysis on secreted IgG.
The cross linking of CD40 is critical for stimulation of B cells. Therefore, you need to coat culture plates with CD40L first, then add B cell. But, from my experience, CD40L feeder cells are more efficient in terms of CD40 cross lining as compared to coated CD40L. I do not recommend to just use soluble CD40L. At least you should coat plates with soluble CD40L.
Hi Toshimitsu,
Thanks for your kind reply. Since in ELISPOT assay (memory B cell stimulation), we easily add soluble CD40 and it always work, I thought that soluble CD40 will work here as well. Anyway, thanks for your recommendation re CD40 coating.
CD40L normally works as a trimer. Therefore you need to crosslink CD40, using anti-CD40 Ab or molecules such as CD40L-CD8 fusion protein plus anti-CD8 Ab, to get better proliferation of B cells. This kind of CD40 stimulation, however, soon induces B cell differentiation to plasma cells, which hampers further proliferation of B cells. The clones of 3T3 cells expressing CD40L selected for good B-cell growth should be better for expansion of single B cells, for unknown mechanisms.
Thanks Daisuke for your answer. Actually, I have tried soluble anti bovine CD40 on a population of bovine B cells (not single cell) and it worked. So, I am wondering if for single B cell stimulation, I can coat the plate with the mentioned bovine CD40. If CD40L expressing cell lines are more efficient, I am also wondering if 3T3 ms CD40L cell line is helpful to stimulate bovine B cells. In other word, do I need a bovine CD40L expressing cell line or the 3T3 ms CD40L is fine?
I don't know if murine or human CD40L can stimulate bovine CD40 on bovine B cells. You will see if you will try it. All I know is mouse CD40L on 3T3 cells can stimulate human B cells to proliferate.
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