Dear colleagues,
I'm trying to isolate B cells from mouse spleen and subsequently
culture them about 24 hours. My question is, do I need stimulants (eg. IL-4, CD40L) other than basic culture conditions (RPMI / FBS 10% / p/s)for primary B cell? I'm trying to utilize Miltenyi Biotec B cell isolation kit.
Thanks for your help in advance.
Hi Joo Sung,
It depends what you want to do. Cultured in RPMI/10% FBS/Penstrep (+2-Me), the purified naive follicular B cells will remain viable (>98%) for 24 h without anything else. In that window, if you add IL-4 and CD40L or anti-CD40 mAb you will activate the cells, but very few (
Hi Joo Sung,
What is your downstream application? What do you want to do with these cells after 24h?
Using the Miltenyi Biotech B cell isolation kit will remove most of the activated B cells, plasma cells and B1 cells. However, upon isolation your splenic B cell fraction still contains Follicular B cells (Fo B) and Marginal Zone B cells (MZB) as well as transitional B cells.
It's a heterogenous mixture of B cell subsets. This is important if you want to isolate protein or RNA from these cells, because many transcripts and/or proteins are differentially expressed in Fo B and MZB (and transitional B cells).
In addition, these cell types also react differently to different mitogens/antigens.
Depending on your experimental read out, you should consider to perform cell sorting to get purified follicular B cells or purified Marginal Zone B cells.
Best
Wolfgang
Hi Wolfgang,
Thanks for your comment. I'm trying to examine cytokines such as IL6, Ifna and Tnfa. I think I should consider to further isolate the cells. Thanks a lot.
Regards,
Joo sung.
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