Approximate yield of B Cells from PBMCs

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TheyCallMeAce
TheyCallMeAce's picture
Approximate yield of B Cells from PBMCs

Hello,

I am interested in isolating B cells from a PBMC sample using the B Cell Isolation Kit II from Miltenyi Biotec. The source is donor whole blood, I believe. I am wondering whether to use MS Columns with MiniMACs or LS Columns with MidiMACs.

A secondary question would be if I can freeze the B cells, or keep them in culture for a few days. My goal would be to do functional assays on them, and I'd need at least a couple of million B cells per assay.

Thanks,
Ace

marcus muench
marcus muench's picture
 I would follow the miltenyi

 I would follow the miltenyi protocol for the isolation.  They should give you a good idea of what cell numbers can be processed with the tow different columns.

You always take a loss when freezing cells, so avoid that if you can.  There are many different B-cell culture methods out there for making antibodies, isotype switching etc.  Generally some combination of feeder cells, conditioned media (T-cell) and cytokines is used.  What you should do depends on what you are going after.

xm833
xm833's picture
I agree that you should start

I agree that you should start with the Miltenyi protocol as it is written and work from there. I will add that I've found a surprising amount of diversity in the B cell recovery from PBMC from different donors. I'm not sure whether this has to do with innate differences in the donors or is ude to colelction/storage/etc. I'd recommend trying to get a fresh a sample as possible and purifying rapidly.

Freezing will cause losses, and the quality of the cells coming out of the freeze can be poor (they may not respond in assays the way freshly isolated cells do). There are indeed published methods for culturing B cells and as noted above they require some substantial efforts (cytokines, conditioned media, feeders) which can introduce variability-so expect some time working out conditions. B cells tend not to do as well in culture as T cells. Depending upon what you need to do it is possible to efficiently transduce with retroviruses, and you can potentially immortalize with EBV or other protocols.