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.

Figure 1

Flowchart of Ab labeling and magnetic sorting steps for sorting non-GC and GC B cells.

Figure 2

Representative flow cytometry plots. (a–b) A successful sort (a), and an unsuccessful sort (b). Presorted total splenocytes are shown in black (a, upper left). Blue and red gates indicate GC and non-GC populations, respectively, as indicated by GL7 and Fas (CD95) staining (a, upper left). Post-sort purity for GCs (a, upper right) and non-GCs (a, lower left) are indicated as the percentage of total events in the single-cell gate. For non-GCs, B220 and IgD staining confirm that GL7⁻ Fas⁻ cells are mature B2 cells (a, lower right). Frequency of CD11c⁺ dendritic cells and CD138⁺ plasma cells are indicated as percentage of GL7⁻ Fas⁻ non-GCs (b, top) from an unsuccessful GC sort (b, bottom).