I'm looking for a protocol to measure the proliferation of primary murine B cells following 72 hours of stimulation with anti-IgM, anti-CD40, LPS, and CD40 & IgM. I've tried CFSE (5 & 2.5uM - 5 & 15min 37C) staining but do not get well defined peaks of proliferation making it difficult to analyze the data. Additionally, the staining doesn't appear to be uniform throughout the sample. Recently, I've tried utilizing BrdU (BioLegend) for the last 16-24 hours of culture. I've tried both 10uM and 40uM and obtained better results with 40uM. However, I only get about 5% positive for incorporation of BrdU. This is extremely reduced compared to what I see with my CFSE cultures. Any advice/suggestions would be greatly appreciated.
Cheers,
Sarah
Dear Sarah Sumner
Please see my B cell proliferation protocol using CFSE staining attached. I think that there is some room for optimization as your CFSE concentration can be titrated down to 1 uM.
In addition, please have a look at publications from my postdoc for the complete B cell activation protocol with different stimuli.
Article MYSM1-dependent checkpoints in B cell lineage differentiatio...
Article USP44 is dispensable for normal hematopoietic stem cell func...
Article Regulation of B Lymphocyte Development by Histone H2A Deubiq...
Good luck with your experiment!
All the best & kind regards,
Michael
Thank you so much for the protocol Michael Förster !! I will definitely try that in the upcoming weeks. I've been trying to use a 96 well plate format, have you had any experience with this? I do like the idea of seeding at a higher density in a larger well I'm just not sure that I'll be able to isolate enough B cells to test all my conditions. Thank you!
Thank you for the response Peter W. Krenn ! I've also tried adding BrdU for the entire 72 hour culture but have more cell death that way. As far as CFSE staining, I haven't rested the cells on ice before washing so I'll definitely be trying that in the future.
Thank you both for the suggestions/advice!
Dear Sarah Sumner
Concerning your question, I have not tested CFSE staining in 96 well plates, as particularly the quenching step (addition of FCS) becomes impossible. I always refrained using 96 well plates, as quenching is essential to keep your cells alive (as overexposure to CFSE will affect viability negatively). Unfortunately, it's hard to get enough B cells, especially, if one of your experimental groups is a KO with B cell deficiency, but you may improve your results by combining splenic and lymph node B cells.
Let me know about your experience with the protocol and drop me a line, if you have additional questions.
All the best & kind regards,
Michael
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