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

Nat Protoc. 2011 Jun 9;6(7):953-60. doi: 10.1038/nprot.2011.344.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Antigens, Differentiation / immunology
  • B-Lymphocytes / cytology
  • B-Lymphocytes / immunology*
  • Biotinylation
  • CD11c Antigen / immunology
  • Germinal Center / cytology*
  • Immunoglobulin D / immunology
  • Immunomagnetic Separation / methods*
  • Leukosialin / immunology
  • Mice
  • Mice, Inbred C57BL
  • Spleen / cytology


  • Antigens, Differentiation
  • CD11c Antigen
  • Immunoglobulin D
  • Leukosialin
  • antigen GL7