title: Magnetic-based purification of untouched mouse germinal center B cells for ex vivo manipulation and biochemical analysis.
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.
authors: Program on Inflammatory Disease Research, Infectious and Inflammatory Disease Center, Sanford-Burnham Medical Research Institute, La Jolla, California, USA.
authors: Matthew H
authors: MH
authors: Cato
authors: Irene W
authors: IW
authors: Yau
authors: Robert C
authors: RC
authors: Rickert
mesh_terms: D000818:Animals; D000943:Antigens, Differentiation / Q000276:immunology; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology*; D019921:Biotinylation; D039521:CD11c Antigen / Q000276:immunology; D018858:Germinal Center / Q000166:cytology*; D007072:Immunoglobulin D / Q000276:immunology; D018189:Immunomagnetic Separation / Q000379:methods*; D051917:Leukosialin / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D013154:Spleen / Q000166:cytology
chemical_list: D000943:Antigens, Differentiation; D039521:CD11c Antigen; D007072:Immunoglobulin D; D051917:Leukosialin; C096712:antigen GL7