Dear Lauro,

I just briefly wanted to send you some considerations for the design of your experiment. I have worked with a knock out mouse that had a severely diminished B cell compartment. Isolating B cells from these mice with a negative selection kit never yielded enough cells to set up proper in vitro cultures (with comparable cell numbers).

For that reason I had to switch to a positive selection kit (I used B220+ from Miltenyi, which is pretty much interchangeable with the CD19+ kit). However, when I did that the cells were pre-activated (compared to negatively selected B cells when stained for activation markers like CD80, CD86, CD54 or MHCII), which isn't a big surprise as you massively pour on antibodies. The other problem I encountered was that my class switch recombination assay (with IgM-BCR crosslink as positive control) was difficult on the read out side (as it was extremely hard to find antibody combinations to detect class switched B cells in FACS for IgM stimulated cells). I suppose the B cells which were covered with antibodies (B220 & IgM) could not be properly stained for flow cytometry for sterical hindrance. There was no issue in detecting IgG in culture supernatants by ELISA, which convinced me that my assay worked. When I stimulated with LPS, IL-4, anti-CD40 antibodies or combinations thereof, I had none of the aforementioned problems.

I hope those tips help you to design your experiment. For further consideration check out:

Article MYSM1-dependent checkpoints in B cell lineage differentiatio...

All the best,

Michael

As Michael said, your yields will be low with negative selection. However, as you want to measure the effect of activation, I would try negative selection anyway. I would suggest a ficoll and then the CD3 kit from miltenyi works fine. You would have to take into consideration that the purity of negatively-selected cells is lower than positively selected ones, and may be around 60% before optimization of the Ab concentration.

You can measure that with a flow cytometer anyway.

Best,

D

Hi Lauro,

When looking at activation/functionality of cell subsets post isolation/enrichment, it is better to use negative selection. As Michael and Daniel mentioned, you will have to compromise on the yield but this way you can avoid your cells getting activated during positive isolation. If you are working with wt mice, roughly 50% of splenocytes should be B cells (https://www.stemcell.com/media/files/wallchart/WA10011-Frequencies_Percentages_Mouse_Immune_Cell_Types.pdf).

I know Miltenyi is big in Europe (I did my PhD there and was using their kits for isolating T cells). One disadvantage when working with column based kits is that the column itself could activate your cells of interest. StemCell Technologies (based in Canada) have B cell isolation kits which are column free and use negative selection (links below). Maybe you could try these. I have been using their kits for isolation of both mouse and human T/B/DC/monocyte subsets and would highly recommend them. The process usually takes 10-20 minutes depending on the kit you get. They also provide sample sizes free of charge so you could test them out.

https://www.stemcell.com/easysep-mouse-b-cell-isolation-kit.html

https://www.stemcell.com/easysep-mouse-pan-b-cell-isolation-kit.html

Hi everyone!

Thanks a lot for your suggestions and precious inputs. I will go for negative selection then. I'll do a pilot experiment with the Pan B Cell Isolation Kit II from Miltenyi, as we already have it in the lab.

All the best,

Lauro

Things could be simple, you could either use CD19 or B220 to stain splenic cells to get a pure B population (adding markers for specific subsets are equally ok), by adding DUMP staining marker to get rid of DC, Macrophages., then Pan B Cell iso kit from Mitenyi works fine. I tested both for single-cell or bulk PCR, ELISPOT, FACS, even differentiaiton in feeder and the results are the same.