Abstract
Detailed biochemical analysis of unmanipulated germinal center (GC) B cells has not been achieved. Previously, we designed and used a simple, economical and new magnetic bead separation scheme for the purification of 'untouched' mature GC and non-GC B cells from the spleens of immunized mice and reported the first biochemical assessment of the signaling cascades that contribute to cyclin D stability and GC B cell proliferation. Here we provide a detailed protocol for the method we used, which involves preparing single-cell suspension from the spleens of immunized mice, followed by labeling of nontarget cells with biotinylated antibodies specific for CD43, CD11c and IgD (for GC enrichment) or GL7 (for non-GC enrichment); these steps are followed by cell depletion using standard magnetic bead technology. This protocol can yield GC and non-GC B cells with purities exceeding 90%. The sorting process can be carried out in ∼1 h and provides a population of GC B cells of sufficient purity and quantity to allow ex vivo manipulation, including biochemical and genetic analysis as well as cell culture.
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Acknowledgements
We thank the Sanford-Burnham Medical Research Institute vivarium and flow cytometry core staff for their dedicated research support. We also thank the members of the Rickert lab for many fruitful discussions. This work was supported by the US National Institutes of Health (AI041649 to R.C.R.).
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M.H.C. designed the GC and non-GC sorting protocol, including initial troubleshooting and proof of concept trials, authored the abstract and all sections of the introduction, and generated Figure 2. I.W.Y. further optimized the protocol, improving on yield and purity; authored the materials, procedure and timing sections; and generated Figure 1. The troubleshooting and anticipated results sections and Table 1 were coauthored by M.H.C. and I.W.Y. R.C.R. edited the manuscript.
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Cato, M., Yau, I. & Rickert, R. Magnetic-based purification of untouched mouse germinal center B cells for ex vivo manipulation and biochemical analysis. Nat Protoc 6, 953–960 (2011). https://doi.org/10.1038/nprot.2011.344
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DOI: https://doi.org/10.1038/nprot.2011.344
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