Hi! Anyone use FACS sorting for isolation of mouse splenic B-cells without using dynabeads? any protocol for culture of isolated primary B-cells invitro?
Google is flooded with Miltenyi protocols like science never existed before!
Hi, I just started sorting and cultivation of rabbit B-cells. I think it should work with mice as well. I found the most useful information in this patent:
http://www.google.com/patents/US20130084637
Hi, I think Muller's suggested protocol covered everything. Just some remind, if you intended to culture the primary B cells after sorting, keep it in sterile condition is very important, including the machine. After sorted the cells, try to reduce the volume of the buffer and increase the FBS might help to increase the survive rate. Good luck.
See protocol 1 in the attached as an example of "old school" approach. No antibodies needed, but I would not call it the easiest approach for B-cell isolation.
See DeFuria et al PNAS 2013 from my lab where we sorted follicular and marginal zone B cells from BL/6 spleen. Culture conditions should be in that paper too, but are basically RPMI/10%FCS/P/S.
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