Magnetic-based purification of untouched mouse germinal center B cells for ex vivo manipulation and biochemical analysis

摘要

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.